Oligonucleoside linkages containing adjacent nitrogen atoms

ABSTRACT

Novel compounds that mimic and/or modulate the activity of wild-type nucleic acids. In general, the compounds contain a selected nucleoside sequence wherein the nucleosides are covalently bound through linking groups that contain adjacent nitrogen atoms.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/039,979, filed Mar.30, 1993 now abandoned, which in turn is a continuation-in-part ofPCT/US92/04294 filed on May 21, 1992, and of Ser. No. 07/903,160 filedon Jun. 24, 1992 now abandoned, which is a continuation-in-part of Ser.No. 07/703,619 filed on May 21, 1991 now U.S. Pat. No. 5,378,825, whichis a continuation-in-part of U.S. Ser. No. 07/566,836 filed Aug. 13,1990 which is now U.S. Pat. No. 5,223,618 issued on Jun. 29, 1993, and acontinuation-in-part of U.S. Ser. No. 07/558,663 filed 27 Jul., 1990which is now U.S. Pat. No. 5,138,045 issued on Aug. 11, 1992.

This application also is related to the subject matter disclosed andclaimed in the following patent applications filed herewith by thepresent inventors: the patent application entitled "OligonucleosideLinkages Containing Adjacent Oxygen and Nitrogen Atoms"; the patentapplication entitled "Backbone Modified Oligonucleotide Analogs AndPreparation Thereof Through Radical Coupling"; and the patentapplication entitled "Backbone Modified Oligonucleotide Analogs AndPreparation Thereof Through Reductive Coupling". Each of these patentapplications are assigned to the assignee of this application and areincorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofnuclease resistant oligonucleotide analogs which are useful fortherapeutics, diagnostics and as research reagents. Oligonucleotideanalogs are provided having modified linkages replacing thephosphorodiester bonds that serve as inter-sugar linkages in wild typenucleic acids. Such analogs are resistant to nuclease degradation andare capable of modulating the activity of DNA and RNA.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are effected by proteins. Proteins, either actingdirectly or through their enzymatic functions, contribute in majorproportion to many diseases in animals and man.

Classical therapeutics generally has focused upon interactions withproteins in an effort to moderate their disease causing or diseasepotentiating functions. Recently, however, attempts have been made tomoderate the production of proteins by interactions with the molecules(i.e., intracellular RNA) that direct their synthesis. Theseinteractions have involved hybridization of complementary "antisense"oligonucleotides or certain analogs thereof to RNA. Hybridization is thesequence-specific hydrogen bonding of oligonucleotides oroligonucleotide analogs to RNA or to single stranded DNA. By interferingwith the production of proteins, it has been hoped to effect therapeuticresults with maximum effect and minimal side effects.

The pharmacological activity of antisense oligonucleotides andoligonucleotide analogs, like other therapeutics, depends on a number offactors that influence the effective concentration of these agents atspecific intracellular targets. One important factor foroligonucleotides is the stability of the species in the presence ofnucleases. It is unlikely that unmodified oligonucleotides will beuseful therapeutic agents because they are rapidly degraded bynucleases. Modification of oligonucleotides to render them resistant tonucleases therefore is greatly desired.

Modification of oligonucleotides to enhance nuclease resistancegenerally has taken place on the phosphorus atom of the sugar-phosphatebackbone. Phosphorothioates, methyl phosphonates, phosphoramidates andphosphorotriesters have been reported to confer various levels ofnuclease resistance. Phosphate-modified oligonucleotides, however,generally have suffered from inferior hybridization properties. See,e.g., Cohen, J. S., ed. Oligonucleotides: Antisense Inhibitors of GeneExpression, (CRC Press, Inc., Boca Raton Fla., 1989).

Another key factor is the ability of antisense compounds to traverse theplasma membrane of specific cells involved in the disease process.Cellular membranes consist of lipid-protein bilayers that are freelypermeable to small, nonionic, lipophilic compounds and are inherentlyimpermeable to most natural metabolites and therapeutic agents. See,e.g., Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological andantiviral effects of natural and modified oligonucleotides in culturedmammalian cells have been well documented. It appears that these agentscan penetrate membranes to reach their intracellular targets. Uptake ofantisense compounds into a variety of mammalian cells, including HL-60,Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has beenstudied using natural oligonucleotides and certain nuclease resistantanalogs, such as alkyl triesters and methyl phosphonates. See, e.g.,Miller, et al., Biochemistry 1977, 16, 1988; Marcus-Sekura, et al., Nuc.Acids Res. 1987, 15, 5749; and Loke, et al., Top. Microbiol. Immunol.1988, 141, 282.

Often, modified oligonucleotides and oligonucleotide analogs areinternalized less readily than their natural counterparts. As a result,the activity of many previously available antisense oligonucleotides hasnot been sufficient for practical therapeutic, research or diagnosticpurposes. Two other serious deficiencies of prior art compounds designedfor antisense therapeutics are inferior hybridization to intracellularRNA and the lack of a defined chemical or enzyme-mediated event toterminate essential RNA functions.

Modifications to enhance the effectiveness of the antisenseoligonucleotides and overcome these problems have taken many forms.These modifications include base ring modifications, sugar moietymodifications and sugar-phosphate backbone modifications. Priorsugar-phosphate backbone modifications, particularly on the phosphorusatom, have effected various levels of resistance to nucleases. However,while the ability of an antisense oligonucleotide to bind to specificDNA or RNA with fidelity is fundamental to antisense methodology,modified phosphorus oligonucleotides have generally suffered frominferior hybridization properties.

Replacement of the phosphorus atom has been an alternative approach inattempting to avoid the problems associated with modification on thepro-chiral phosphate moiety. For example, Matteucci, Tetrahedron Letters1990, 31, 2385 disclosed the replacement of the phosphorus atom with amethylene group. However, this replacement yielded unstable compoundswith nonuniform insertion of formacetal linkages throughout theirbackbones. Cormier, et al., Nucleic Acids Research 1988, 16, 4583,disclosed replacement of phosphorus with a diisopropylsilyl moiety toyield homopolymers having poor solubility and hybridization properties.Stirchak, et al., Journal of Organic Chemistry 1987, 52, 4202 disclosedreplacement of phosphorus linkages by short homopolymers containingcarbamate or morpholino linkages to yield compounds having poorsolubility and hybridization properties. Mazur, et al., Tetrahedron1984, 40, 3949, disclosed replacement of a phosphorus linkage with aphosphonic linkage yielded only a homotrimer molecule. Goodchild,Bioconjugate Chemistry 1990, 1, 165, disclosed ester linkages that areenzymatically degraded by esterases and, therefore, are not suitable forantisense applications.

The limitations of available methods for modification of the phosphorusbackbone have led to a continuing and long felt need for othermodifications which provide resistance to nucleases and satisfactoryhybridization properties for antisense oligonucleotide diagnostics andtherapeutics.

OBJECTS OF THE INVENTION

It is an object of the invention to provide oligonucleotide analogs fordiagnostic, research, and therapeutic use.

It is a further object of the invention to provide oligonucleotideanalogs capable of forming duplex or triplex structures with, forexample, DNA.

It is a further object to provide oligonucleotide analogs havingenhanced cellular uptake.

Another object of the invention is to provide oligonucleotide analogshaving greater efficacy than unmodified antisense oligonucleotides.

It is yet another object of the invention to provide methods forsynthesis and use of oligonucleotide analogs.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification and theappended claims.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that mimic and/ormodulate the activity of wild-type nucleic acids. In general, thecompounds contain a selected nucleoside sequence which is specificallyhybridizable with a targeted nucleoside sequence of single stranded ordouble stranded DNA or RNA. At least a portion of the compounds of theinvention has structure I: ##STR1## wherein: L₁ --L₂ --L₃ --L₄ is CH₂--NR₁ --NR₂ --CH₂ or NR₁ --NR₂ --CH₂ --CH₂ ;

R₁ and R₂ are the same or different and are H; alkyl or substitutedalkyl having 1 to about 10 carbon atoms; alkenyl or substituted alkenyl2 to about 10 carbon atoms; alkynyl or substituted alkynyl having 2 toabout 10 carbon atoms; alkaryl, substituted alkaryl, aralkyl, orsubstituted aralkyl having 7 to about 14 carbon atoms; alicyclic;heterocyclic; a reporter molecule; an RNA cleaving group; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of anoligonucleotide;

B_(x) is a nucleosidic base;

n is an integer greater than 0;

Q is O, S, CH₂, CHF or CF₂ ;

X is H, OH, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide.

The compounds of the invention generally are prepared by couplingpreselected 3'-functionalized and 4'-functionalized nucleosides and/oroligonucleotides under conditions effective to form the above-noted L₁--L₂ --L₃ --L₄ linkages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic, synthetic scheme describing preparation oflinkages of the invention through use of dimethoxytrityl-protectedintermediates;

FIG. 2 is a schematic, synthetic scheme describing preparation oflinkages of the invention through use of phthalimido-protectedintermediates;

FIG. 3 is a schematic, synthetic scheme describing preparation ofcompounds 106-108;

FIG. 4 is a schematic, synthetic scheme describing preparation ofcompounds 114-118;

FIG. 5 is a schematic, synthetic scheme describing preparation ofcompounds 10 and 27-29;

FIG. 6 is a schematic, synthetic scheme describing preparation ofcompound 31;

FIG. 7 is a schematic, synthetic scheme describing preparation ofcompound 23;

FIG. 8 is a schematic, synthetic scheme describing preparation ofcompound 47;

FIG. 9 is a schematic, synthetic scheme describing preparation ofcompounds 42-45;

FIG. 10 is a schematic, synthetic scheme describing preparation ofcompound 54;

FIG. 11 is a schematic, synthetic scheme describing preparation ofcompound 62;

FIG. 12 is a schematic, synthetic scheme describing preparation ofcompound 14;

FIG. 13 is a schematic, synthetic scheme describing preparation ofcompound 9

FIG. 14 is a schematic, synthetic scheme describing preparation ofcompounds 15 and 20;

FIG. 15 is a schematic, synthetic scheme describing preparation ofcompounds 14 and 36-38;

FIG. 16 is a schematic, synthetic scheme describing preparation ofcompound 58;

FIG. 17 is a schematic, synthetic scheme describing preparation ofcompound 66;

FIG. 18 is a schematic, synthetic scheme describing preparation ofcompound 49;

FIG. 19 is a schematic, synthetic scheme describing preparation ofcompound 72;

FIG. 20 is a schematic, synthetic scheme describing preparation ofcompounds 77, 81, and 85;

FIG. 21 is a schematic, synthetic scheme describing preparation ofcompound 89.

DETAILED DESCRIPTION OF THE INVENTION

The term "nucleoside" as used in connection with this invention refersto a unit made up of a heterocyclic base and its sugar. The term"nucleotide" refers to a nucleoside having a phosphate group on its 3'or 5' sugar hydroxyl group. Thus nucleosides, unlike nucleotides, haveno phosphate group. "Oligonucleotide" refers to a plurality of joinednucleotide units formed in a specific sequence from naturally occurringbases and pentofuranosyl groups joined through a sugar group by nativephosphodiester bonds. This term refers to both naturally occurring andsynthetic species formed from naturally occurring subunits.

The compounds of the invention generally can be viewed as"oligonucleotide analogs", that is, compounds which function likeoligonucleotides but which have non-naturally occurring portions.Oligonucleotide analogs can have altered sugar moieties, altered basemoieties or altered inter-sugar linkages. For the purposes of thisinvention, an oligonucleotide analog having non-phosphodiester bonds,i.e., an altered inter-sugar linkage, is considered to be an"oligonucleoside." The term "oligonucleoside" thus refers to a pluralityof nucleoside units joined by linking groups other than nativephosphodiester linking groups. The term "oligomers" is intended toencompass oligonucleotides, oligonucleotide analogs or oligonucleosides.Thus, in speaking of "oligomers" reference is made to a series ofnucleosides or nucleoside analogs that are joined via either naturalphosphodiester bonds or other linkages, including the four atom linkersof this invention. Although the linkage generally is from the 3' carbonof one nucleoside to the 5' carbon of a second nucleoside, the term"oligomer" can also include other linkages such as 2'-5' linkages.

Oligonucleotide analogs also can include other modifications consistentwith the spirit of this invention, particularly modifications thatincrease nuclease resistance. For example, when the sugar portion of anucleoside or nucleotide is replaced by a carbocyclic moiety, it is nolonger a sugar. Moreover, when other substitutions, such a substitutionfor the inter-sugar phosphorodiester linkage are made, the resultingmaterial is no longer a true nucleic acid species. All such compoundsare considered to be analogs. Throughout this specification, referenceto the sugar portion of a nucleic acid species shall be understood torefer to either a true sugar or to a species taking the structural placeof the sugar of wild type nucleic acids. Moreover, reference tointer-sugar linkages shall be taken to include moieties serving to jointhe sugar or sugar analog portions in the fashion of wild type nucleicacids.

This invention concerns modified oligonucleotides, i.e., oligonucleotideanalogs or oligonucleosides, and methods for effecting themodifications. These modified oligonucleotides and oligonucleotideanalogs exhibit increased stability relative to their naturallyoccurring counterparts. Extracellular and intracellular nucleasesgenerally do not recognize and therefore do not bind to thebackbone-modified compounds of the invention. In addition, the neutralor positively charged backbones of the present invention can be takeninto cells by simple passive transport rather than by complicatedprotein-mediated processes. Another advantage of the invention is thatthe lack of a negatively charged backbone facilitates sequence specificbinding of the oligonucleotide analogs or oligonucleosides to targetedRNA, which has a negatively charged backbone and will repel similarlycharged oligonucleotides. Still another advantage of the presentinvention is it presents sites for attaching functional groups thatinitiate cleavage of targeted RNA.

The modified internucleoside linkages of this invention preferablyreplace naturally-occurring phosphodiester-5'-methylene linkages withfour atom linking groups to confer nuclease resistance and enhancedcellular uptake to the resulting compound. Preferred linkages havestructure CH₂ --R_(A) --NR₁ --CH₂, CH₂ --NR₁ --R_(A) --CH₂, R_(A) --NR₁--CH₂ --CH₂, or NR₁ --R_(A) --CH₂ --CH₂ where R_(A) is NR₂. R_(A) alsocan be O. Those skilled in the art will recognize that moieties whereinR_(A) is NR₂ and O will undergo many common reactions.

The compounds of the invention generally are prepared by couplingpreselected 3'-functionalized and 4'-functionalized nucleosides and/oroligonucleotides under conditions effective to form the above-noted L₁--L₂ --L₃ --L₄ linkages. In certain embodiments, the compounds of theinvention are prepared by intermolecular reductive coupling. In otherembodiments, the compounds of the invention are prepared byintermolecular radical addition reactions.

In the reductive coupling methods, a 3'-formyl nucleoside oroligonucleotide synthon is reacted with a 5'-hydrazino nucleoside oroligonucleotide synthon. In other embodiments, a 5'-formyl synthon isreacted with a 3'-methylhydrazino synthon. In still further embodiments,linkages having structure CH═N--R_(A) --CH₂, CH₂ --CH═N--R_(A), CH₂--R_(A) --N═CH, or R_(A) --N═CH--CH₂ where R_(A) is NR₁ are formed bycoupling synthons having structures II and III: ##STR2## wherein: Z₁ andY₂ are selected such that

(i) Z₁ is C(O)H and Y₂ is CH₂ R_(A) NH₂ ; or

(ii) Z₁ is CH₂ R_(A) NH₂ and Y₂ is C(O)H;

(iii) Z₁ is CH₂ C(O)H and Y₂ is R_(A) NH₂ ; or

(iv) Z₁ is R_(A) NH₂ and Y₂ is H(O)CCH₂ ;

Y₁ is aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxy- methyl, aryl-substituted imidazolidino,aminohydroxylmethyl, ortho-methylamino-benzenethio, methylphosphonate,methyl-alkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide,an oligonucleoside, or a hydroxyl-protected or amine-protectedderivative thereof;

Z₂ is hydroxyl, aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxy-methyl, aryl-substituted imidazolidino,aminohydroxylmethyl, ortho-methylamino-benzenethio, methylphosphonate,methyl-alkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide,an oligonucleoside, or a hydroxyl-protected or amine-protectedderivative thereof;

B_(x1) and B_(x2) are, independently, nucleosidic bases;

Q₁ and Q₂ are, independently, O, S, CH₂, CHF or CF₂ ; and

X₁ and X₂ are, independently, H, OH, alkyl, substituted alkyl, alkarylor aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide.

The radical addition reactions can be divided in two steps. The firststep involves generation of an initial radical, which undergoes thedesired reaction. The second step involves removal of the radical fromthe reaction before the occurrence of an intervening, undesired reactionsuch as cross coupling. In certain embodiments, the linkages of theinvention are prepared by providing donor synthons having structure IVaor Va: ##STR3## wherein Z_(1a) and Y_(2a) have structure CH₂ --R₈ or R₈where R₈ is a radical generating group, generating a radical centered atZ_(1a) or Y_(2a) and then forming a 3'-5' linkage by reactingradical-bearing donor synthons IVa and Va, respectively, with acceptorsynthons Vb and IVb: ##STR4## wherein either Z_(1b) and Y_(2b) havestructure NR₁ --N═CH₂ or CH₂ --NR₁ --N═CH₂.

B_(x1) and B_(x2) can be nucleosidic bases selected from adenine,guanine, uracil, thymine, cytosine, 2-aminoadenosine or5-methylcytosine, although other non-naturally occurring species can beemployed to provide stable duplex or triplex formation with, forexample, DNA. Representative bases are disclosed in U.S. Pat. No.3,687,808 (Merigan, et al.), which is incorporated herein by reference.

Q₁ and Q₂ can be S, CH₂, CHF CF₂ or, preferably, O. See, e.g., Secrist,et al., Abstract 21, Synthesis and Biological Activity of4'-Thionucleosides, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

X₁ and X₂ are, independently, H, OH, alkyl, substituted alkyl, alkarylor aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide. It isintended that the term "alkyl" denote branched and straight chainhydrocarbyl residues, including alkyl groups having one or more ³ Hand/or ¹⁴ C atoms. It is preferred that X is H or OH, or, alternativelyF, O-alkyl or O-alkenyl, especially where Q is O. Preferred alkyl andalkenyl groups have from 1 to about 10 carbon atoms.

Y₁ and Z₂ can be aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxymethyl, aryl-substituted imidazolidino,aminohydroxylmethyl, ortho-methylaminobenzenethio, methylphosphonate,methyl-alkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide,an oligonucleoside, or a hydroxyl-protected or amine-protectedderivative thereof. In addition, Z₂ can be hydroxyl. Preferably, Y₁ is aprotected hydroxymethyl group or a nucleoside or oligonucleosideattached by, for example, a phosphodiester-5'-methylene linkage or someother four atom linking group, and Z₂ is a protected hydroxyl group or anucleoside or oligonucleoside attached by, for example, aphosphodiester-3'-hydroxyl linkage or some other four atom linkinggroup.

It is preferred that the oligonucleotide analogs of the inventioncomprise from about 5 to about 50 subunits having the given structure(i.e., n═5-50). While each subunit of the oligonucleotide analogs canhave repetitive structure I, such need not be the case. For example, thesubunits can have alternating or more random structures.

Reductive Coupling

The linkages of the invention can be formed by selecting a 3'-C-formylderivatized compound as the upstream synthon and a 5'-hydrazinoderivatized compound as the downstream synthon. Coupling then iseffected to provide, for example, a dinucleoside having an iminelinkage. The imine linked compound can be incorporated directly into anoligomer and/or can be reduced to a corresponding hydrazino linkedspecies. Reduction of imine linked dinucleosides either as thedinucleoside or as a dinucleoside moiety in an oligomer with sodiumcyanoborohydride yields the corresponding hydrazino linked compounds.Hydrazino linked compounds can be N-alkylated to yield correspondingN-alkylamino linkages. 3'-C-formyl derivatized nucleosides can be formedvia several synthetic pathways. The presently preferred method utilizesa radical carbonylation of the corresponding 3'-deoxy-3'-iodonucleoside. The iodo compound is treated with CO,2,2'-azobisisobutrylonitrile (AIBN), and tris(trimethylsilyl) silane(TTMS). Alternately, 3'-C-formyl derivatized compounds can besynthesized from either a 3'-deoxy-3'-cyano sugar or nucleoside. Both5'-C-formyl (also identified as 5'-aldehydo) and 3'-C-formyl group canbe blocked in a facile manner utilizing o-methylamino-benzenthiol as ablocking group. The 5'-and 3'-C-formyl groups can be deblocked withsilver nitrate oxidation.

An alternate method of 3'-C-formyl nucleoside synthesis employs1naturally -O-methyl-3'-deoxy-3'-O-methylaminobenzenethiol-5'-O-trityl-β-D-erythro-pento furanoside, which serves as aprecursor for any 3'-deoxy-3'-C-formyl nucleoside. The1-0-methyl-3'-deoxy-3'-O-ethyl aminobenzenethiol-5'-O-trityl-β-D-erythro-pentofuranoside is reacted with anappropriate base utilizing standard glycosylation conditions and thendeblocked to yield the nucleoside. In yet another method, a3'-deoxy-3'-cyano nucleoside is prepared from either the corresponding3'-deoxy-3'-iodo nucleoside or by glycosylation with1-O-methyl-3'-deoxy-3'-O-cyano-5'-O-trityl-β-D-erythro-pentofuranoside.

Resulting dinucleosides from any of the above described methods, linked,for example, by hydrazines can be protected by a dimethoxytrityl groupat the 5'-hydroxyl and activated for coupling at the 3'-hydroxyl withcyanoethyldiisopropyl-phosphite moieties. These dimers can be insertedinto any desired sequence by standard, solid phase, automated DNAsynthesis utilizing phosphoramidite coupling chemistries. The protecteddinucleosides are linked with the units of a specified DNA sequenceutilizing normal phosphodiester bonds. The resulting oligonucleotideanalog or oligomer has a "mixed" backbone containing both phosphodiesterlinkages and four atoms linkages of the inventions. In this manner, asequence-specific 15-mer oligonucleotide can be synthesized to haveseven hydroxylamine, hydrazine or other type linked dinucleosidesattached via alternating phosphodiester linkages. Such a structure willprovide increased solubility in water compared to fully modifiedoligomers, which may contain linkages of the invention.

Oligonucleosides containing a uniform backbone linkage can besynthesized by use of CPG-solid support and standard nucleic acidsynthesizing machines such as Applied Biosystems Inc. 380B and 394 andMilligen/Biosearch 7500 and 8800s. The initial nucleoside (number 1 atthe 3'-terminus) is attached to a solid support such as controlled poreglass or polystyrene beads. In sequence specific order, each newnucleoside is attached either by manual manipulation or by the automatedsynthesizer system. In the case of a methylenehydrazine linkage, therepeating nucleoside unit can be of two general types--a nucleoside witha 5'-protected aldehydic function and a 3'-deoxy-3'-C-hydrazinomethylgroup, or a nucleoside bearing a 5'-deoxy-5'-hydrazino group and aprotected 3'-deoxy-3'-C-formyl group. In each case, the conditions whichare repeated for each cycle to add the subsequent sequence required baseinclude: acid washing to remove the 5'-aldehydo protecting group;addition of the next nucleoside with a 3'-methylenehydrazino group toform the respective hydrazone connection; and reduction with any of avariety of agents to afford the desired methylene-hydrazine linked CPG-or polystyrene-bound oligonucleosides. One such useful reducing agent issodium cyanoborohydride.

A preferred method is depicted in FIG. 1. This method employs a solidsupport to which has been bound a downstream synthon having a protected5' site. Preferably, the 5' site of said synthon is protected with DMT.Thereafter, the 5' site of the synthon is liberated with mild acid,washed, and oxidized to produce an intermediate product. In onepreferred method, the aldehyde derivative reacts withN,N-diphenylethylene diamine to produce an intermediary product, a5'-diphenylimidazolidino protected synthon. In a more preferred methodthe 5'-diphenylimidazolidino protected synthon is directly loaded on thesupport. With either method, the intermediary product can besubsequently deblocked to provide a synthon with a nucleophilic 5'position.

An upstream synthon having a protected 5'-aldehyde group, such as a5'-diphenylimidazolidino protected 3'-deoxy-3'-C-hydrazine base, iscoupled with the bound downstream synthon under acidic conditions. Theintermediate hydrazine derivative is then reduced by sodiumcyanoborohydride to furnish a dinucleoside linked through a hydrazinomoiety. Thereafter, the cycle can be repeated by the addition of anupstream synthon, followed by acid/base deprotection to create apolymeric synthon of a desired sequence containing modified inter-sugarlinkages. In some preferred embodiments of this invention, the upstreamsynthon is a 5'-FMOC protected 3'-C-methylenehydrazino base.

One preferred process employs a diphenylethyldiamine adduct(1,3-disubstituted imidazolidino) to protect the electrophilic center ofthe downstream synthon during attachment to the solid support. Moffatt,et al., J. Am. Chem. Soc. 1968, 90, 5337. The downstream synthon can beattached to a solid support such as a controlled pore glass support orother suitable supports known to those skilled in the art. Attachmentcan be effected via standard procedures. Gait, M. J., ed.,Oligonucleotide Synthesis, A Practical Approach (IRL Press 1984).Alternatively, the protected bound nucleoside can be oxidized bystandard oxidizing procedures. Bound downstream synthons preferably arereacted with hydrazine to produce a Schiff's base, which can be reduced.

A further method of synthesizing uniform backbone linkedoligonucleosides is depicted in FIG. 2 (R'=hydroxyl protecting group,R"=phthalimido). This method also employs a solid support to which hasbeen bound a downstream synthon with a protected 5' site. The 5' sitepreferably is protected with a phthalimido group. The 5' site of thedownstream synthon is liberated with, for example, t-butylammoniumfluoride (TBAF). The hydrazine group at the 5' position of the upstreamsynthon also is protected with a phthalimido group to yield a5'-phthalimido protected 3'-deoxy-3'-C-formyl nucleoside, which isreacted with the downstream synthon. Deprotection at the 5' positionliberates the next 5'-hydrazino reaction site. The cycle is repeatedwith the further addition of upstream synthon until the desired sequenceis constructed. Each nucleoside of this sequence is connected withmethylenehydrazine linkages. The terminal nucleoside of the desiredoligonucleoside preferably is added to the sequence as a 5'-O-silylblocked 3'-deoxy-3'-C-formyl nucleoside. The methylenehydrazine linkedoligonucleoside are then removed from the support. If a hydrazine linkedoligonucleoside is desired, the methylenehydrazine linkages are reducedwith sodium cyanoborohydride. Alternately reduction can be accomplishedwhile the methylenehydrazine linked oligonucleoside is still connectedto the support.

Radical Coupling

The radical-based methods of the invention generally involve "nonchain"processes. In nonchain processes, radicals are generated bystoichiometric bond homolysis and quenched by selective radical-radicalcoupling. It has been found that bis(trimethylstannyl)-benzopinacolateand bis(tributylstannyl)benzopinacolate (see Comprehensive OrganicSynthesis: Ed. by B. M. Trost & J. Fleming, Vol. 4, pp 760)--persistentradicals--can be used to enhance the radical-radical coupling and reducecross-coupling. It will be recognized that a persistent radical is onethat does not react with itself at a diffusion-controlled rate.Hillgartner, et al., Liebigs. Ann. Chem. 1975, 586, disclosed that onthermolysis (about 80° C.) pinacolate undergoes homolytic cleavage togive the suspected persistent radical (Ph₂ C.OSnMe₃), which stays inequilibrium with benzophenone and the trimethylstannyl radical (Me₃Sn.). It is believed that the Me₃ Sn. radical abstracts iodine fromradical precursors such as 3'-deoxy-3'-iodo nucleosides or5'-deoxy-5'-iodo nucleoside derivatives to give 3' or 5' nucleosideradicals. The nucleoside radicals then add to, for example, hydrazinoacceptors such as 3'- or 5'-deoxy-3' or 5'-methylenehydrazine nucleosidederivatives to give a dimeric nucleoside containing a dephosphointernucleoside linkage.

The concentration of the persistent radical is an important variable inthese reactions because at high concentrations the initial radical canbe trapped by coupling prior to addition, and at low concentrations theadduct radical can begin to telomerize. It is believed that a 3 molarequivalent excess of pinacolate provides satisfactory results for suchcouplings. The efficiency of radical reactions are highly dependent onthe concentration of the reagents in an appropriate solvent. Preferably,the solvent contains, for example, benzene, dichlorobenzene,t-butylbenzene, t-butyl alcohol, water, acetic acid, chloroform, carbontetrachloride, and mixtures thereof. The solvent should contain acombined concentration of about 0.1 to about 0.4 moles/liter of radicalprecursor and acceptor, preferably about 0.1 to about 0.2 moles/liter,more preferably about 0.2 moles/liter. It has been found that bestresults are obtained using benzene solutions containing about 0.2moles/liter of radical precursor and acceptor.

As exemplified in FIG. 3, chain elongation in a 5' to 3' sense can beachieved generally by refluxing a 0.2-0.4 molar solution of5'-deoxy-5'-iodo-3'-N-protected nucleoside 102 (R_(A) =NR₁, R'=hydroxylprotecting group, R"=phthalimido),3'-deoxy-3'-methylenehydrazino-5'-protected nucleoside 101, andbis(trimethylstannyl)-benzopinacolate in benzene under argon for 8 h toyield dimeric nucleoside 103 (L₁ --L₂ --L₃ --L₄ =NR₁ --NH--CH₂ --CH₂) in35% yield after purification by silica gel chromatography. This dimercan be methylated following standard procedures by, for example,utilizing aqueous formaldehyde (20% solution) to furnish N-alkylated 104(L₁ =L₂ =N--CH₃). Further hydrazinolysis of and formylation of theproduct will furnish 3'-imine 105 (R"=N═CH₂), which is ready for anotherround of radical coupling. Thus, coupling this dimer with bifunctionalnucleoside 102 will provide trimer 106 (L_(1a) --L_(2a) --L_(3a)--L_(4a) =NR₁ --NH--CH₂ --CH₂, n=1). In a similar manner, trimer 106 canundergo another round of coupling to furnish a tetrameric nucleoside.Repetitive coupling of this type will provide an oligomer of desiredlength. Chain elongation can be terminated at any time during thedescribed method. For example, coupling of dimer ether 105 with a5'-deoxy-5'-iodo-3'-protected nucleoside will furnish trimer 107 (L_(2a)=N--CH₃), which could be N-methylated (L₂ =N--CH₃) and deblocked at its3' and 5' ends to yield deprotected trimer 108 (R'=R"=H).

As exemplified in FIG. 4, chain elongation in a 3' to 5' sense can beachieved generally by refluxing a concentrated (0.2 to 0.3 molar)solution of 3'-deoxy-3'-iodo-51-N-protected nucleoside 109, 5'-deoxy-5'(methylenehydrazine!-3 '-protected nucleoside 110, andbis(trimethylstannyl)benzopinacolate in benzene under argon for 8 h toyield dimeric nucleoside 111 (L₁ --L₂ --L₃ --L₄ =CH₂ --NH--NR₁ --CH₂,R'=hydroxyl protecting group, R"=phthalimido) in 45% yield afterpurification by silica gel chromatography. Dimer 111 can be methylatedfollowing standard Procedures to furnish N-alkylated 112 (L₁ =L₂ =N--CH₃). Subsequently, hydrazinolysis of 112, followed by formylation of theproduct will furnish 113 (R'=N═CH₂) Dimer 113 can undergo another roundof radical coupling with 109 to yield trimeric nucleoside 114 (L_(1a)--L_(2a) --L_(3a) --L_(4a) =CH₂ --NH--NR₁ --CH₂, n=1). The lattercompound could be N-methylated to yield 115 (L_(1A) =L₂ =N--CH₃). Arepetitive set of reactions such as hydrazinolysis, formylation, andcoupling would result in an oligomer of desired length containingmodified backbones. Chain elongation can be terminated at any point bycoupling with 3'-deoxy-3'-iodo-5'-protected nucleoside. For example, thelatter compound will couple with 113, and the product on methylation (L₂=N--CH₃) and deblocking will furnish trimeric nucleoside 116 (R'=R"=H).

A more random type of elongation can be effected by deblocking andiodinating nucleoside 104 selectively at its 3' end to producenucleoside 117a (R'=O-blocking group, R"=I). Coupling of 117a with 110via 3'-elongation furnishes trimeric nucleoside 118a (L₂ =N--CH₃ andL_(2a) =NH). Methylation and complete deblocking of 118a provides 118b(R'=R"=OH and L₂ =N--CH₃). Alternatively, deblocking and iodinatingnucleoside 112 selectively at its 5' end produces nucleoside 119a (R'=I,R"=O-blocking group). Coupling of 119a with 101 via 5'-elongationfurnishes trimeric nucleoside 118a. Methylation and complete deblockingprovides 118b. ##STR5##

A wide range of achiral and neutral oligonucleosides containing mixedbackbones can be prepared by these "random" methods. Backbone complexitycan be enhanced further by selectively incorporating phosphodiesterlinkages to, for example, increase water solubility. (See, e.g., Example97). Also, coupling 5'-O-dimethyoxytritylated dimeric nucleoside 117b(R'=ODMTr, R"=OH) or trimeric nucleoside 118c (R'=ODMTr, R"=OH, L₂=N--CH₃ and L_(2a) =NH) to CPG via a succinyl linker (see, e.g., NucleicAcids Research 1990, 18, 3813) provides CPG-bound compounds that can beused as 3'-terminal units for automated synthesis. The placement of suchdimeric or trimeric oligonucleosides at the 3'-end of an antisenseoligomer will result in the protection of the antisense molecules fromattack by 3'-exonucleases typically found in human serum.

In summary, the chain elongation methods of the invention requireessentially two types of nucleoside building blocks. The first is amonofunctional nucleoside with one reactive group for coupling at 3' or5' end and an appropriate blocking group at the remaining end. Thesecond is a bifunctional nucleoside which has a reactive coupling groupat 3' or 5 end and a protected reactive coupling group at the other end.

The methods of the invention can be modified for use with eithersolution-phase or solid-phases techniques. For example, the compounds ofthe invention can be synthesized using controlled pore glass (CPG)supports and standard nucleic acid synthesizing machines such as AppliedBiosystems Inc. 380B and 394 and Milligen/Biosearch 7500 and 8800s. Eachnew nucleoside is attached either by manual manipulation or by automatedtechniques.

A wide variety of protecting groups can be employed in the methods ofthe invention. See, e.g., Beaucage, et al., Tetrahedron 1992, 12, 2223.In general, protecting groups render chemical functionality inert tospecific reaction conditions, and can be appended to and removed fromsuch functionality in a molecule without substantially damaging theremainder of the molecule. Representative hydroxyl protecting groupsinclude t-butyldimethylsilyl (TBDMSi), t-butyldiphenylsilyl (TBDPSi),dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and other hydroxylprotecting groups as outlined in the above-noted Beaucage reference.

FIG. 5 illustrates certain abbreviations used for blocking groups inother of the Schemes. FIG. 5 further shows the synthesis of 3'-O-aminoand 3'-O-methyleneamino nucleosides via a Mitsunobu reaction utilizingN-hydroxylphthalimide and methylhydrazine to generate an--O--NH₂ moietyon a sugar hydroxyl. The -O--NH₂ group can then be derivatized to a-O-methyleneamino moiety. These reactions are exemplified in Examples25-27.

The reactions of Examples 25-27 represent improved syntheses of3'-O--NH₂ nucleosides. In forming--O--NH₂ moieties on sugars, it istheoretically possible to displace a leaving group, as for instance atosyl group, with hydroxylamine. However, Files, et al., J. Am. Chem.Soc. 1992, 14, 1493 have shown that such a displacement leads to apreponderance of -NHOH moieties and not to the desired--O--NH₂ moieties.Further, the reaction sequence of Examples 25-27 represents an improvedsynthesis compared to that illustrated in European Patent Application 0381 335. The synthetic pathway of that patent application requires theuse of a xylo nucleoside as the starting material. Xylo nucleosides areless readily obtainable than the ribonucleoside utilized in Examples25-27.

FIG. 6 illustrates the conversion of a 4'-aldehydo nucleoside to a5'-aldehydo nucleoside. This reaction is exemplified in Example 31. FIG.7 illustrates the generation of a 5'-aldehydo methyl sugar. This isexemplified in Example 29. FIG. 8 illustrates the formation of an5'-iodo nucleoside. Similar methodology is used to generate an activeiodo group on a terminal hydroxyl of a dimeric unit in Scheme XII. InFIG. 8, the iodo nucleoside is further derivatized to a 6'-aldehydonucleoside via an allyl substituted nucleoside. This is exemplified inExamples 46 and 47.

FIG. 9 illustrates a free radical reaction of a -O-methyleneaminonucleoside of FIG. 6 to a 5'-amino 5'-homo nucleoside. This isexemplified in Example 45. FIG. 10 illustrates use of a Mitsunobureaction on a 5'-homo nucleoside to synthesize an oxyaminehomonucleoside, i.e. a 6'-O--NH2 5'-homo nucleoside. This is exemplifiedin Examples 49, 50, and 51. FIG. 11 illustrates N-alkylation of theamino moiety of a 6'-amino-5'-deoxy-5'-homo nucleoside. This isexemplified in Examples 56, 57, and 58. Such N-alkylation is desirablewhere the amino moiety subsequently will be reacted with a thiol moiety.The N-alkylated product of such a reaction exhibits greater stability toacid than does the non-alkylated S--N bond. This is particularly usefulin solid support synthesis wherein acid removal of trityl groups iscommonly practiced. However, for other synthesis, such as solutionsynthesis, this may not be a concern.

FIGS. 12 to 21 illustrate the use of nucleosides for the assembly ofdimeric, trimeric and other, higher order oligonucleosides. In FIG. 12,nucleosides 3 and 31 are joined via an acid catalyzed coupling reactionto form an -O-nitrilomethylidyne linkage between the respective twonucleosides. This is exemplified in Example 32. Dimeric oligonucleoside32 can be reduced to an iminomethylene linkage that, in turn, can bealkylated to a (methylimino)methylene linkage, as exemplified in Example33.

FIG. 13 illustrates the coupling of nucleoside 3 to nucleoside 5. Thisscheme is analogous to FIG. 12 with the exception that in Scheme X athree atom linkage is created whereas in Scheme X a four atom linkage iscreated. Nucleosides 3 and 5 are joined in Step 1 to form an -O-nitrilolinkage that is reduced in Step 2 to an -O-imino linkage. Alkylationoccurs in Step 3 to a -O-methylimino linkage, with final deblocking inStep 4. These steps are exemplified in Example 28. The alkylationreaction in Step 3 is accompanied by deblocking the t-butyldimethylsilylprotecting group at the 5' terminus of the dimer. Advantageous use ofthis deblocking reaction also is utilized in other schemes. Deblockingof the t-butyldiphenylsilyl group used to protect the 3' terminus of thedimer is effected using tetra-n-butylammonium fluoride.

The alkylation step can be used to introduce other, useful, functionalmolecules on the macromolecule. Such useful functional molecules includebut are not limited to reporter molecules, RNA cleaving groups, groupsfor improving the pharmacokinetic properties of an oligonucleotide, andgroups for improving the pharmacodynamic properties of anoligonucleotide. Such molecules can be attached to or conjugated to themacromolecule via attachment to the nitrogen atom in the backbonelinkage. Alternatively, such molecules can be attached to pendent groupsextending from the 2' position of the sugar moiety of one or more of thenucleosides of the marcromolecules. Examples of such other usefulfunctional groups are provided by U.S. patent application Ser. No.782,374, filed Oct. 24, 1991, entitled Derivatized OligonucleotidesHaving Improved Uptake & Other Properties, assigned to the same assigneeas this application, herein incorporated by reference, and in other ofthe above-referenced patent applications.

FIG.14 illustrates a synthetic scheme utilized to prepare dimers,trimers, and other, higher order oligonucleosides having homogenouslinkages between nucleosides. In this scheme, nucleosides 10 and 12 arelinked to form an iminomethylene linkage. Advantageous use of thealkylating-5' terminus deblocking step of FIG. 12 is effected to removethe blocking group at the 5' terminus of the dimeric oligonucleoside 14.Using the iodination reaction of FIG. 8, the dimer is then converted toa 5' terminus iodo intermediate. A further 3'-O-methyleneaminonucleosidic unit 10 then can be added to the dimer to form a trimer,followed by deblocking and alkylation. This reaction sequence can berepeated any number of times to form a higher order oligonucleoside. Theoligonucleoside is deblocked at the 3' terminus.

FIG. 16 illustrates the use of an 1-O-alkyl sugar that is first linkedto a nucleoside. Reduction followed by alkylation and deblocking yieldsan -O-(methylimino)methylene linkage joining the 1-O-alkyl sugar and thenucleoside, as exemplified by Example 34. This structure is then blockedat the 5' terminus, as exemplified by Example 35. The fully blocked,linked sugar-nucleoside structure is then subjected to glycosylation toadd a heterocyclic base to the sugar moiety and thus form a dimericnucleoside structure, as in Example 36. After glycosylation, removal ofthe 5' terminus blocking group and chromatographic separation of α and βanomers, as exemplified by Example 37, yields a dimer. This dimer can befurther elongated as per the procedure of FIG. 14. Examples 39 and 40exemplify the addition of an adenine, cytosine and guanine base to athymidine-methyl sugar dimer to form T-A, T-C and T-G dimers in additionto the T-T dimer of Scheme XII. Examples 41, 42, and 43 exemplify theformation of A-T, A-A, A-C, A-G, C-T, C-A, C-C, C-G, G-T, G-A, G-C andG-G dimers. Each may be further elongated as per the procedures ofScheme XII.

FIG. 16 illustrates the formation of an imino-oxymethylene linkage.Example 48 describes the preparation of the 5'-0-trityl protected xylostarting nucleoside and Example 52 describes the reaction of compound 50with compound 54 to form a dimeric unit. Continuing within FIG. 16, toprepare dimeric units that can be used as solid support building blocks(Example 53), the backbone nitrogen atom is alkylated, followed bysimultaneous removal of both the 5'-O-trityl and the3'-0-(t-butyldiphenylsilyl) protecting groups with trifluoroacetic acid.The 5'-terminus hydroxyl group is blocked with dimethoxytriryl (Example54), followed by forming an active phosphoramidate dimer (Example 55).

FIG. 17 illustrates the preparation of a thiol intermediate and the useof that intermediate with an amino nucleoside to form a S-iminomethylenelinkage (Example 58). As with the reactions of FIG. 16, a dimeric unithaving an active phosphoramidate moiety can be formed. This isexemplified by Examples 59 and 60.

FIG. 19 illustrates the preparation of a nucleoside intermediate andcoupling of that intermediate to a further nucleoside, as exemplified inExample 61, to form a nitrilo-1,2-ethanediyl linkage. This linkage canbe reduced to an imino-1,2-ethanediyl linkage, as exemplified in Example62. Further, in a manner similar to FIGS. 16 and 17, FIG. 19 illustratesthe preparation of an active phosphoramidate species, as exemplified inExamples 63, 64, and 65.

FIG. 20 illustrates the preparation of a 2' substituted nucleoside, asexemplified in Example 66, and conversion of that 2' substitutednucleoside to a further nucleoside having an active linkage formingmoiety (Example 67). Linkage of this 2' substituted nucleoside to afurther nucleoside (Example 68) is followed by conversion to an activephosphoramidate (Example 69). Substitution of the 2' position in amacromolecule of the invention, as noted above, is useful for theintroduction of other molecules, including the introduction of reportermolecules, RNA cleaving groups, groups for improving the pharmacokineticproperties of an oligonucleotide, and groups for improving thepharmacodynamic properties of an oligonucleotide as well as other groupsincluding but not limited to O, S and NH alkyl, aralkyl, aryl,heteroaryl, alkenyl, alkynyl and ¹⁴ C containing derivatives of thesegroups, F, Cl, Br, CN, CF₃, OCF₃, OCN, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃,NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino and substituted silyl.

Further illustrated in Scheme XVIII is the preparation of a carbocyclicnucleoside (Example 70), joining of that carbocyclic nucleoside with afurther nucleoside via a linkage of the invention (Example 71), andformation of an active phosphoramidate (Example 76). A further sequenceof reactions are also illustrated in Scheme XVIII, wherein a carbocyclicnucleoside is derivatized at its 2' positions (Example 73) and convertedto a further nucleoside (Example 74). As with the other reactions ofthis scheme, a dimer is first formed (Example 75), and then derivatizedwith an active phosphoramidate (Example 76). The dimers of this schemehaving a 3' phosphoramidite moiety are used as in FIGS. 17, 14 and 15 tolink the oligonucleosides of the invention to other nucleosides via aphosphodiester, phosphorothioate or other similar phosphate basedlinkage.

FIG. 21 illustrates a further carbocyclic containing, dimericnucleoside. Internucleoside conversion is exemplified in Examples 77 and78, and formation of a dimeric structure is exemplified in Example 79.The dimeric structure of Scheme XIX shows a carbocyclic nucleoside asthe 5' nucleoside of the dimer, while FIG. 20 shows a carbocyclicnucleoside as the 3' nucleoside of the dimer. Use of carbocyclicnucleosides for both nucleoside intermediates, in the manner asdescribed for other of the reaction schemes, results in a dimer having acarbocyclic nucleoside at both the 3' and 5' locations.

Compound 120 illustrates generic structures that are prepared from thenucleosides and oligonucleoside of the previous schemes. Exemplarymacromolecules of the invention are described for both solid support andsolution phase synthesis in Example 81. ##STR6##

The compounds of this invention can be used in diagnostics,therapeutics, and as research reagents and kits. For therapeutic use theoligonucleotide analog is administered to an animal suffering from adisease modulated by some protein. It is preferred to administer topatients suspected of suffering from such a disease an amount ofoligonucleotide analog that is effective to reduce the symptomology ofthat disease. One skilled in the art can determine optimum dosages andtreatment schedules for such treatment regimens.

It is preferred that the RNA or DNA portion which is to be modulated bepreselected to comprise that portion of DNA or RNA which codes for theprotein whose formation or activity is to be modulated. The targetingportion of the composition to be employed is, thus, selected to becomplementary to the preselected portion of DNA or RNA, that is to be anantisense oligonucleotide for that portion.

In accordance with one preferred embodiment of this invention, thecompounds of the invention hybridize to HIV mRNA encoding the tatprotein, or to the TAR region of HIV mRNA. In another preferredembodiment, the compounds mimic the secondary structure of the TARregion of HIV mRNA, and by doing so bind the tat protein. Otherpreferred compounds complementary sequences for herpes, papilloma andother viruses.

It is generally preferred to administer the therapeutic agents inaccordance with this invention internally such as orally, intravenously,or intramuscularly. Other forms of administration, such astransdermally, topically, or intralesionally may also be useful.Inclusion in suppositories may also be useful. Use of pharmacologicallyacceptable carriers is also preferred for some embodiments.

This invention is also directed to methods for the selective binding ofRNA for research and diagnostic purposes. Such selective, strong bindingis accomplished by interacting such RNA or DNA with compositions of theinvention which are resistant to degradative nucleases and whichhybridize more strongly and with greater fidelity than knownoligonucleotides or oligonucleotide analogs.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting,wherein parts and percents are by weight unless otherwise indicated. ForNMR analysis of dimers and other higher oligonucleosides, monomericunits are numbered (e.g., T₁, T₂) from the 5' terminus towards the 3'terminus nucleoside. Thus, the 5' nucleoside of a T-T dimer is T₁ andthe 3' nucleoside is T₂.

General Procedures

Radical Addition Reaction

A suspension of radical precursor (1-3 eq.), radical acceptor (1 eq.),persistent radical (3 eq.) in benzene (0.2-0.4 mol. solution) wascarefully degassed under vacuum (water aspirator) and flushed with argon(3-times). The reaction mixture was heated at 80°-85° C. for 6-12 hunder argon while stirring. The suspension dissolves to give a clearsolution in about 1-2 h. The reaction mixture may change the colorduring the course of heating but remains clear all along. Completion ofthe reaction was judged by the disappearance of radical precursor(detected by TLC) and formation of a polar product. The reaction mixturethen was cooled to room temperature and diluted with ether (five timesthe original volume). The fine suspension was loaded onto a prepackedsilica gel column (30 g of silica per gram of product) and eluted withhexanes (100%) until most of the UV absorbing impurities were removed.The column then was eluted with a hexanes-ether gradient in which theconcentration of ether gradually increased to effect a 10% increase inthe polarity of the solvent. Elution with ether furnished the desiredproduct as homogeneous material. Pooling and evaporation of appropriatefractions generally gave 40-55% yield of the desired dimeric nucleoside.

N-Alkylation of Backbone

To a stirred solution of oligonucleoside (1 eq.; containing CH₂-O-NH-CH₂ linkages) in glacial acetic acid was added an aqueous solutionof HCHO (3-5 eq.; >99% HCHO) in one portion under argon. The clearsolution was stirred for 5-15 min. at room temperature until no morestarting material was detected by TLC. At this point dry NaBH₃ CN (3-6eq.) was added in 3-6 portions under argon at room temperature under awell ventilated fume hood. Care must be taken to cool the reactionmixture if it is over 5 mmol. scale. After addition, the reactionmixture was stirred for 1-2 h at room temperature (until evolution ofgas ceases). Completion of the reaction was detected by TLC, whichindicated formation of a higher product, compared to the startingmaterial. The reaction mixture was concentrated under vacuum and theresidue purified by short column chromatography. Elution with a CH₂ Cl₂→ MeOH gradient (increasing polarity to 10%) furnished the desiredproduct as homogeneous material.

This procedure was applicable to a variety of substituted aldehydeswhich formed Schiff bases with the amino group of the linker. Subsequentreduction gave selective alkylation of the backbone. Heterocyclic orexocyclic amines of the purine/pyrimidine were not affected by thismethod.

Composition Analysis of Modified Oligomers

Incorporation of oligonucleoside containing backbones of the inventioninto antisense molecules was proved by enzymatic tandem hydrolysis ofmodified oligomers using snake-venom phosphodiesterase followed byalkaline phosphatase. In all cases, the identity of dimeric nucleosideswas proven by addition of synthetic sample and comparison on HPLCprofile. The integration of the peaks of HPLC analysis demonstrated thecorrect gross composition of the modified oligomer.

EXAMPLE 1 Synthesis of 5'-deoxy-5'-hydrazino nucleosides

(a) 5'-Deoxy-5'-hydrazinothymidine hydrochloride

To provide 5'-benzylcarbazyl-5'-deoxythymidine, 5'-O-tosylthymidine,Nucleosides & Nucleotides 1990, 9, 89! (1.98g, 5 mmol), benzylcarbazide(4.15 g, 25 mmol), activated molecular sieves (3A, 2 g), and anhydrousdimethylacetamide (100 ml) were stirred together with exclusion ofmoisture at 110° C. (bath temperature) for 16 hours. The products werecooled and concentrated under reduced pressure (bath temperature <50°C.). The residue was purified on a silica gel column (5×45 cm) with CH₂Cl₂ /MeOH (9:1, v/v) as the solvent. The homogeneous fractions werepooled, evaporated to dryness and the foam recrystallized from EtOH toyield 0.7 g (36%) of 5'-benzylcarbazyl-5'-deoxythymidine; mp 201° C.; ¹H NMR (Me₂ SO-d₆) δ 1.79 (s, 3, CH₃), 2.00-2.18 (m, 2, C₂, CH₂), 2.95(t, 2, C₅, CH₂), 3.75 (m, 1, C₄,H), 4.18 (m, 1, C₃,H), 4.7 (brs, 1, O'₂NH), 5.03 (s, 2, PhCH₂), 5.2 (d, 1, C₃,H), 6.16 (t, 1, C₁,H), 7.2-7.4(m, 5, C₆ H₅), 7.6 (s, 1, C₆ H), 8.7 (brs, 1, CH₂ NH), 11.2 (brs, 1, C₃NH).

To provide the hydrochloride salt of 5'-deoxy-5'-hydrazinothymidine as ahygroscopic powder, a mixture of the above carbazate (0.78 g, 2 mmol)and palladium on charcoal (10%, 150 mg) in anhydrous MeOH/HCl (30 ml,2%, HCl by weight) was stirred under an atmosphere of hydrogen at roomtemperature for 1.5 hours. The methanolic solution was filtered throughCelite to remove the catalyst. The filter cake was washed with EtOH(2×25 ml). The filtrate was concentrated under vacuum and the residuewas dried overnight to remove traces of HCl. The yellow residue wasdissolved in methanol (3 ml) and added dropwise to a rapidly stirredsolution of ethyl acetate (150 ml). The filtered precipitate was washedwith ethyl acetate (3×100 ml) and the pale yellow solid was dried undervacuum to yield 0.51 g (88%) of 5'-deoxy-5'-hydrazinothymidinehydrochloride (hygroscopic powder); ¹ H NMR (Me₂ SO-d₆) δ 1.81 (s, 3,CH₃), 2.02-2.22 (m, 2, C_(2') CH₂), 3.2 (m, 2, C₅ CH₂), 3.8, (m, 1,C₄,H), 4.2 (m, 1, C₃, H), 6.17 (t, 1, C₁,H), 7.54 (s, 1, C₆ H), 11.18(brs, 1, C₃ NH), the hydrazino and 3'-OH were masked by H₂ O.

EXAMPLE 2 Synthesis of 5'-O-trityl-1-2,3-dideoxy-3-C-(formyl)-β-D-erythro-pentofuranosyl!-thymine and -uracil

Method A

3'-C-Cyano-3-deoxy-5'-O-tritylthymidine

The following preparation should to be performed under a hood and allprecautions taken not to inhale any of reagent fumes. A suspension of3'-deoxy-3'-iodo-5'-O-tritylthymidine (Verheyden, et al., J. Org. Chem.1970, 35, 2868) (60 g, 0.1 mol), hexamethylditin (36 g, 22.7 ml, 0.11mol), t-butylisocyanide (166 g, 225 ml, 2 mol), and AIBN (1.6 g, 10mmol) in toluene (freshly distilled over Na/benzophenone, 2 lt) wasthoroughly deoxygenated by bubbling argon through the reaction mixturefor 30 min. and then heated at 80° C. for 13 h. The reaction mixture wascooled at 60° C. and AIBN (1.6 g, 10 mmol) was added and heatingcontinued for 24 h. During this period addition of AIBN was repeated for3 times in an identical manner. The reaction mixture was cooled to roomtemperature and transferred on the top of a prepacked silica gel column(1.5 kg, in hexanes) and eluted with hexanes: Et₂ O (100% hexanes → 100%Et₂ O with a 10% gradient change each time using 1 lt of eluent). Mostof the impurities were removed during the gradient elution as non-polarcompounds. Final elution with Et₂ O (2 lt), pooling and evaporation ofappropriate fractions gave two compounds in the order these werecollected. (i) 12.93 g (25%) of 3'-C-Cyano-3'-deoxy-5'-Q-tritylthymidineas white powder (crystallized from toluene/Et₂ O, mp 153°-157° C.); ¹ HNMR (CDCl₃) δ 8.83 (s, 1, NH), 7.04-7.4 (m, 18.5, TrH, C₆ H, and 0.5 ArHfrom toluene), 6.10 (dd, 1, H_(1'), J_(1'),2' =4.1 Hz, J_(1'),2" =7.1Hz), 4.20 (m, 1, H_(4'), J_(3'),4' =8.4 Hz, J_(4'),5' =2.8 Hz),3.33-3.60(m, 3, H_(5'), 5", 3'), 2.68 (m, 1, H_(2'), J_(2'), 2" =13.8Hz), 2.52 (m, 1, H_(2")), 2.28 (s, 1.5, 0.5 CH₃ from toluene), and 1.50(s, 3, CH₃). Anal. Calcd. for C₃₀ H₂₇ N₃ O₄. 0.5 C₇ H₈ (toluene fromcrystallization): C, 74.56; H, 5.79; N, 7.78. Found: C, 74.27; H, 5.78;N, 7.66.

The reaction mixture also gave 4.82 g, (10%) of 1-(3'-C-cyano-2',3'-dideoxy-5'-O-trityl-β-D-threo-entofuranosyl)thymine; 1H NMR (CDC1₃) 68.72 (s, 1, NH), 7.03-7.44 (m, 18.5, TrH, C₆ H, and 0.5 ArH fromtoluene), 6.13 (pseudo t, 1, H_(1'), J_(1'),2' =6.7 Hz, J_('),2" =5.7Hz), 4.09 (m, 1, H_(4'), J_(3'),4' =6.7 Hz, J_(4'),5' =4.9 Hz) , 3.56(m, 2, H_(5'),5", 3.28 (m, 1, H_(3'), J_(3'),2' =8.2 Hz, J_(3'), 2" 5.2Hz), 2.70 (m, 1, H_(2'), J_(2'), 2" =14 Hz), 2.28 (s, 1.5, CH₃ fromtoluene) and 1.60 (s, 3, CH₃). Anal. Calcd. for C₃₀ H₂₇ N₃ O₄. 0.5 C₇ H₈(toluene from crystallization): C, 74.56; H, 5.79; N, 7.78. Found: C,74.10; H, 5.74; N, 7.52.

Epimerization: To a suspension of 1-(3'-C-Cyano-2 ',3'-di-deoxy-5'-O-trityl-β-D-threo-pentofuranosyl)thymine (0.30 g, 0.61mmol) in methanol (20 ml) was added dropwise a 1N solution of NaOMeuntil the pH of solution reached ≈9. The resulting mixture was heated toreflux for 20 h. The solution was cooled (0° C.) and neutralized with 1NHCl/MeOH and evaporated under reduced pressure. The residue was purifiedas described above to furnish 0.185 g (62%) of3'-C-cyano-3'-deoxy-5'-O-tritylthymidine. (A synthesis for3'-deoxy-3'-C-cyano-5'-O-tritylthymine was reported in TetrahedronLetters 1988, 29, 2995. This report suggested3'-deoxy-3'-C-cyano-5'-O-tritylthymine is formed as a single product,however, we found a mixture of threo- and erythro-3'-cyano isomers areproduced. (see, Bankston, et al., J. Het. Chem. 1992, 29, 1405. By theabove epimerization, the xylo component of this mixture is converted tothe compound of interest, 3'-deoxy-3'-C-cyano-5'-O-tritylthymine.)

3'-Deoxy-3'-C-formyl-5'-O-tritylthymine

DIBAL-H (1M in toluene, 50 ml, in 5 portions over a period of 5 h) wasadded to a stirred solution of 3'-C-cyano-3'-deoxy-5'-O-tritylthymidine(9.92 g, 20 mmol) in dry THF (10 ml) under argon at 0° C. The solutionwas stirred at room temperature for 1 h and cooled again to 0° C. MeOH(25 ml) was added dropwise to the cold solution while stirring and aftercomplete addition the solution was brought to room temperature. Asaturated aqueous Na₂ SO₄ solution (11 ml) was added to the reactionmixture and stirred for 12 h. Powdered anhydrous Na₂ SO₄ (30 g) wasadded to the reaction mixture and suspension was stirred for 30 min. Thesuspension was filtered and residue was thoroughly washed with MeOH:CH₂Cl₂ (1:9 v/v) until all of the product was washed off. The filtrateswere combined and concentrated under vacuum, to furnish a gummy residue.The residue was purified by silica gel chromatography using CH₂ Cl₂:MeOH (100% CH₂ Cl₂ → 9:1, v/v) for elution to obtain 5.45 g (55%) of3'-deoxy-3'-C-formyl-5'-O-tritylthymine as a white foam. ¹ H NMR (CDCl₃)δ 9.61 (d, 1, CHO, J_(3'),3" U₁ 1.5 Hz), 8.44 (s, 1, NH), 7.46 (s, 1, C₆H), 7.17-7.45 (m, 15, TrH), 6.04 (pseudo t, 1, H^(1'), J_(1'),2' =5.3Hz, J_(1'),2" =6.6 Hz), 4.31 (m, 1, H_(1'), J_(4'),5' =3.3 Hz, J_(3'),4'=7 Hz), 3.28-3.55 (m, 3, H_(5'),5",3'), 2.69 (m, 1, H_(2')), 2.28 (m, 1,H_(2")), 1.48 (s, 3, CH₃). Anal. Calcd. for C₃₀ H₂₈ N₂ O₅.H₂ O: C,70.03; H, 5.88; N, 5.44. Found: C, 70.40; H, 6.00; N, 5.33.

1- 3-Deoxy-3-C-(formyl)-5-O-trityl-β-D-erythro-pentofuranosyl!uracil

To a stirred solution of 3'-cyano-2', 3'-dideoxy-5'-O-trityluridine(0.96 g, 2 mmol), (prepared in a manner equivalent to that of thethymidine analog above) in dry THF (20 ml) under argon, was added asolution of DIBAL-H in toluene (Aldrich) (1M, 4 ml) at -10° C. over aperiod of 10 min. After 30 mins the reaction was quenched with MeOH (5ml) at -10° C. The mixture was further stirred at ambient temperaturefor 30 mins and diluted with CH₂ Cl₂ (25 ml) before concentrating undervacuum. This process was repeated with CH₂ Cl₂ (3×25 ml) in order toremove the residual THF. The residue was purified by flashchromatography on silica gel (25 g). Elution with CH₂ Cl₂ (9:1, v/v) andcrystallization from CH₂ Cl₂ /MeOH gave 5'-O-trityl-3'-C-formyl-2',3'-dideoxyuridine (0.53 g, 53%); mp 100° C.; 1H NMR (CDCl₃) δ 2.25-2.8(m, 2, CH₂), 3.4 (m, 1, C₃,H), 3.45-3.6 (m, 2, C₅, CH₂), 4.37 (m, 1,C₄,H), 5.4 (d, 1, C₅ H), 6.1 (m, 1, C₁,H), 7.2-7.4 (m, 15, C₆ H₅), 7.81(d, 1, C₆ H), 7.95 (br s, 1, NH), 9.61 (s, 1, HC=O).

Method B

1- 3-deoxy-3-C-(formyl)-5-O-trityl-β-D-erythro-pentofuranosyl!thymine

1-Methyl-5-O-(t-butyldiphenylsilyl)-2,3-dideoxy-3-C-(formyl)-D-erythro-pentofuranosewas obtained as an oil in 90% yield using the DIBAL-H reduction of1-methyl-5-(t-butyldiphenylsilyl)-2,3-dideoxy-3-C-cyano-D-erythro-pentofuranoseas described in Tetrahedron 1900, 44, 625. The 3-C-formyl group isderivatized to the oxime with methoxyamine. The oxime blockedintermediate was glycosylated with silyated thymine to give an α and βmixture of the title compound. After deblocking, the β anomer comparesto that prepared via method A.

Method C

1- 3-deoxy-3-C-(formyl)-5-O-trityl-β-D-erythro-pentofuranosyl! -uraciland -thymine

A mixture of 3'-deoxy-3'-iodo-5'-O-tritylthymidine (0.59 g, 4 mmol),tris(trimethylsilyl) silane (2.87 g, 1.2 mmol), AIBN (12 mg, 0.072mmol), and toluene (20 ml) were mixed in a glass container and saturatedwith argon (bubbling at room temperature). The glass vessel was insertedinto a stainless steel pressure reactor, and pressurized with carbonmonoxide (80 psi), closed and heated (90° C., bath) for 26 hrs. Thereaction mixture was cooled (0° C.) and the CO was allowed to escapecarefully (under the fume hood). The product was purified by flashcolumn chromatography on silica gel (20 g). Elution with EtOAc:Hexanes(2:1, v/v) and pooling the appropriate fractions furnished 0.30 g (61%)of the title compound as a foam.

A radical carbonylation of 2', 3'-dideoxy-3'-iodo-5'-trityluridine in asimilar manner gives the 3'-C-formyl uridine derivative.

EXAMPLE 3 Synthesis of methylenehydrazone linked (3'-CH═NH--NH--CH₂-5'), methylenehydrazine linked (3'-CH₂ --NH--NH--CH₂ -5') andmethylene(dimethylhydrazo) linked (3'--CH₂ --N(CH₃)--N(CH₃)--CH₂ -5')dinucleosides

3'-De(oxyphosphinico)-3-methylene(hydrazone)!-5'-O-tritylthymidylyl-(31'- 5' )-5'-deoxythymidine

A mixture of 3'-deoxy-3'-C-formyl-5'-O-tritylthymidine, 0.645 g, 1.30mmol), 5'-deoxy-5'-hydrazinothymidine hydrochloride (0.397 g, 1.36 mmol)in dry CH₂ Cl₂ /MeOH/AcOH (20 ml/10 ml/0.5 ml) was stirred for 30 min atroom temperature. The solvent was evaporated under vacuum and thehydrazone intermediate was analyzed by ¹ H NMR (DMSO-d₆) δ 1.1 (br s, 2NH), 8.3 (s, 1, C═N--NH), 7.5-7.74 (m, 17, Tr H, 2C₆ H), 6.8 (1d, 1t, 1,HC═N, two isomers), 6.0-6.1 (2m, 2, H_(4')), 5.30 (br t, 1, OH), 3.8-4.2(3m, 3, H_(3'), 2 H_(4')), 3.0-3.3 (m, 5, 2H_(5'),5", H_(3')), 2.0-2.4(m, 4, 2H_(2'), 2) , 1.5 and 1.7 (2s, 6, 2 CH₃).

3'-De(oxyphosphinico)-3'-methylene(dimethylhydrazo)!-5'-O-tritylthymidylyl-(3'→5')-5'-deoxythymidine

The above hydrazone dimer was dissolved in AcOH (10 ml) and to this wasadded small portions of NaBH₃ CN (4×0.12 g, 7.74 mmol) while stirring atroom temperature for 30 min. The solution was stirred for an additional15 min before the addition of aqueous HCHO (20%, 3.9 ml, 26 mmol), NaBH₃CN (3.9 mmol), and ACOH (10 ml). The suspension was further stirred for15 min. and solution evaporated under vacuum. The residue wascoevaporated with MeOH (3×25 ml) to give the methylenehydrazo dimer; ¹ HNMR (CDCl₃) δ 6.8-7.8 (m, 15, TrH, 2 C₆ H), 6.12 (m, 2, 2H_(1')), 4.20((m, 1, T2H_(3')), 4.05 (m, 1, T2H_(4')), 3.89 (m, 1, T1 H_(4')), 3.80(s, 6, 2 OCH₃), 3.21-3.53 (m, 2, T1 H_(5'),5"), 2.11-2.75 (m, 10, T2H_(5'5") H, T1 H_(3"), T1 H_(3'), T1 T2 H_(2'2")), 2.26 (s, 6, 2N--CH₃),1.88 and 1.49 (2s, 6, 2 CH₃), and other protons.

3'-De(oxyphosphinico)-3'-5methylene(dimethylhydrazo)!-thymidylyl-(3'→5')-5'-deoxythymidine

The above hydrazine dimer was then stirred with 37% aqueous HCl (1 ml)in MeOH (25 ml) at room temperature for 24 h. The resulting mixture wasneutralized with NH₄ 0H (pH≈8) and evaporated to dryness. The residuewas purified by reverse phase HPLC (supelcostl LC18, 5 m, H₂ O: CH₃ CNgradient) to furnish 0.61 g of the title methylene(dimethylhydrazine)linked dimer (89%). ¹ H NMR (90° C., DMSO-d₆ +1 drop of D₂ O) δ 7.66 and7.43 (2s, 2, 2 C6H), 6.02 (pseudo t, 1, T2 H_(1'), J_(1'),2' =7.2 Hz,J_(1'),2" =7.7 Hz), 5.96 (pseudo t, 1, T1 H_(1'), J_(1'),2' =5.6 H_(Z),J_(1'),2" =6.2 Hz), 4.12 (m, 1, T2 H_(3')), 3.90 (m, 1, T2 H_(4')), 3.71(m, 1, T1 H_(4')), 3.61 (m, 2, T1 H_(5'),5"), 2.4-2.8 (m, 5, T2H_(5'),5", T1 H_(3"), T1 H_(3"),T1 H_(')) 2.29 (2s, 6, 2 N--CH₃), 2.12(m, 4, 2H_(2'),2"), 1.76 and 1.74 (2s, 6, 2 CH₃). Anal. Calcd. for C₂₃H₃₄ N₆ O₈, H₂ O: C, 51.10, H, 6.71; N, 15.54. Found: C, 51.05; H, 6.68;N, 15.54. MS FAB m/z 523 (M+H)⁺.

EXAMPLE 4 Synthesis of methylene(dimethylhydrazine) linked (3'-CH₂--N(CH₃) --N(CH₃) --CH₂ -5')5'-dimethoxytrityl-3'-β-cyano-ethoxydiisopropylphosphoramiditedinucleosides

3'-De(oxyphosphinico)-3'-methylene(dimethylhydrazo)!-thymidylyl-5'-O-(dimethoxytriphenylmethyl)-(3→5')-3'-O-(β-cyanoethyl-N-diisopropylaminophosphiryl)thymidine

The methylene(dimethylhydrazine) dimer of Example 3 wasdimethyoxytritylated following the standard procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, to furnish a homogenous foam ¹ H NMR (CDCl₃) δ 6.8-7.8 (m,20, DMTr, 2H₆), 6.12 (m, 2, 2H_(1')), 4.20 (m, 1, T₂ H_(3')), 4.05 (m,1, T₂ H_(4')), 3.89 (m, 1, T₁ H_(4')), 3.80 (s, 6, 2 OCH₃ of DMTr),3.21-3.53 (m, 2, T₁ H_(5'5")), 2.11-2.75 (m, 9, T₁ H_(5'5"), H_(3"), T₁H_(3') 2H_(2'2")), 2.26 (2s, 6, 2 N--CH₃) and 1.88 and 1.49 (2s, 2, C₅CH₃) ! which on phosphitylation via the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, provided a 65% yield of the title compound. ¹ H NMR (CDCl₃)δ 6.14 (m, 1, T2 H_(1')), 6.01 (m, 1, T1 H_(1')), 3.80 (s, 6, 2 O CH₃),2.23 (m, 6, 2 N--CH₃), 1.78 and 1.45 (2s, 6, 2 CH₃), and other protons.³¹ P NMR (CDCl₃) δ 149.43 and 148.85 ppm.

EXAMPLE 5 Synthesis of intermittent methylene(dimethyhydrazine) (3'-CH₂--NCH₃ --NCH₃ --CH₂ -5') linked oligonucleosides

CPG-bound thymidine (or any other nucleoside that is to become the3'-terminal base) was placed in an Applied Biosystems, Inc. (ABI) column(250 mg, 10 micromoles of bound nucleoside) and attached to an ABI 380Bautomated DNA Synthesizer. The standard, automated (computer controlled)steps utilizing phosphoramidite chemistries are employed to place themethylenehydrazine thymidine dimer into the sequence at any desiredlocation.

EXAMPLE 6 Synthesis of 5'-O-phthalimido nucleosides

5'-O-Phthalimidothymidine

To a stirred solution of thymidine (24.22 g, 0.1 mol),N-hydroxyphthalimide (21.75 g, 0.13 mol), triphenylphosphine (34 g, 0.13mol) in dry DMF (400 ml) was added diisopropylazodicarboxylate (30 ml,0.15 mol) over a period of 3 h at O° C. After complete addition thereaction mixture was warmed up to room temperature and stirred for 12 h.The solution was concentrated under vacuum (0.1 mm, <40° C.) to furnishan orange-red residue. The residual gum was washed several times withEt₂ O and washing were discarded. The semi-solid residue was suspendedin EtOH (500 ml) and heated (90° C.) to dissolve the product. On cooling30.98 g (80%) of 5'-O-phthalimidothymidine was collected in 3-crops aswhite crystalline material, mp 233°-235° C. (decomp.); H NMR (DMSO-d₆) δ11.29 (s, 1, NH), 7.85 (m , 4, ArH), 7.58 (s, 1, C₆ H), 6.20 (t, 1,H_(1'), J_(1'),2' =7.8 Hz, J_(1'),2" =6.5 Hz), 5.48 (d, 1, OH_(3')),4.36 (m, 3, H_(4'),5',5"), 4.08 (m, 1, H_(3')), 2.09-2.13 (m, 2,H_(2'),2"), and 1.79 (s, 3, CH₃). Anal. Calcd. for C₁₈ H₁₇ O₇ N₃. 0.7 H₂O: C, 54.05; H, 4.64; N, 10.51. Found: C, 53.81; H, 4.25; N, 10.39.

2'-deoxy-5'-O-phthalimidouridine

An analogous reaction on 2'-deoxyuridine gave the corresponding2'-deoxy-51-O-phthalimidouridine; mp 241°-242° C.

EXAMPLE 7 Synthesis of5'-O-phthalimido-3'-O-(t-butyldiphenyl-silyl)thymidine and2'-deoxy-5'-O-phthalimido-3'-O-(t-butyldiphenylsilyl)uridine

3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine

A mixture of 5'-O-phthalimidothymidine (8.54 g, 22 mmol),t-butyldiphenylsilylchloride (6.9 ml, 26.5 mmol), imidazole (3.9 g, 57.3mmol) and dry DMF (130 ml) was stirred at room temperature for 16 hunder argon. The reaction mixture was poured into ice-water (600 ml) andthe solution was extracted with CH₂ Cl₂ (2×400 ml). The organic layerwas washed with water (2×250 ml) and dried (MgSO₄). The CH₂ Cl₂ layerwas concentrated to furnish a gummy residue which on purification bysilica gel chromatography (eluted with EtoAc:Hexanes; 1:1, v/v)furnished 12.65 g (92%) of3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine as crystallinematerial (mp 172°-173.5° C.). ¹ H NMR (DMSO-d₆) δ 11.31 (s, 1, NH), 7.83(m, 4, ArH), 7.59 (m, 4, TBDPhH), 7.51 (s, 1, C₆ H), 7.37-7.45 (m, 6,TBDPhH), 6.30 (dd, 1, H_(1'), J_(1'),2' =8.8 Hz, J_(1'),2", =5.6 Hz),4.55 (m, 1, H_(4')), 4.15 (m, 1, H_(3')), 3.94-4.04 (m, 2, H_(5'),5"),2.06-2.13 (m, 2, H_(2'),2"), 1.97 (s, 3, CH₃), 1.03 (s, 9, C(CH₃)₃).Anal. Calcd. for C₃₄ H₃₅ O₇ N₃ Si: C, 65.26; H, 5.64; N, 6.71. Found: C,65.00; H, 5.60; N, 6.42.

3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidouridine

An analogous reaction of 2'-deoxy-5'-O-phthalimidouridine will give thecorresponding3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidouridine.

EXAMPLE 8 Synthesis of 5'-O-amino nucleoside

5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine

To a stirred solution of3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine (10 g, 16 mmol) indry CH₂ Cl₂ (100 ml) was added methylhydrazine (1.3 ml, 24 mmol) underargon at room temperature and solution stirred for 12 h. The solutionwas cooled (0° C.) and filtered. The white residue was washed with CH₂Cl₂ (2×25 ml) and combined filtrates were evaporated to furnish gummyresidue. The residue on purification by silica gel column chromatography(elution with CH₂ Cl₂ :MeOH, 98:2, v/v) furnished 7.03 g (89%) of5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine that crystallized fromCH₂ Cl₂ /MeOH mp 141°-143° C. ¹ H NMR (DMSO-d₆) δ 11.29 (s, 1, NH),7.42-7.62 (m, 11, TBDPhH, C₆ H), 6.25 (dd, 1, H_(1'), J_(1'),2' =8.4 Hz,J_(1'),2",=6.3 Hz), 6.02 (s, 2, NH₂), 4.35 (m, 1, H_(4')), 4.04 (m, 1,H_(3')), 3.34-3.51 (m, 2, H_(5'), 5"), 2.04 (m, 2, H_(2'), 2"), 1.73 (s,3, CH₃), 1.03 (s, 9, C(CH₃)₃). Anal. Calcd. for C₂₆ H₃₃ O₅ N₃ Si: C,63.00; H, 6.71; N, 8.48. Found: C, 62.85; H, 6.67; N, 8.32.

EXAMPLE 9 Synthesis of (3'-CH═N--O--CH₂ -5') linked oligonucleoside (anoxime linked dimer)

3'-De(oxyphosphinico)-3'-(methylidynenitrilo)-thymidylyl-(3'→5)-thymidine

A mixture of 3'-deoxy-3'-C-formyl-5'-O-tritylthymine (0.99 g, 2 mmol),5'-amino-3'-O-(t-butyldiphenylsilyl) thymidine (0.99 g, 2 mmol) and ACOH(0.3 ml) in dry CH₂ Cl₂ (20 ml) was stirred for 1 h at room temperature.The solvent was evaporated under vacuum and the crude blocked3'-de(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3'→5')-3'-(t-butyl-diphenylsilyl)thymidineproduct was dissolved in THF (20 ml). A THF solution of nBu₄ NF (1M, 5ml) was added to the stirred reaction mixture at room temperature. After1 h solution was purified by silica gel chromatography (elution with CH₂Cl₂ :MeOH; 99:4, v/v) to furnish 3'-deblocked dimer. The dimer wasdissolved in anhydrous MeOH (50 ml) and to this a MeOH/HCl solution(0.14M, 2.5 ml) was added. The reaction mixture was stirred at roomtemperature for 15 h. Anhydrous pyridine (10 ml) was added to the abovesolution and solvents were evaporated to dryness to furnish crude oximedimer. The crude product was purified by silica gel chromatography(elution with CH₂ Cl₂ :MeOH; 92:8, v/v) to furnish the title compound,3'-De(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3→5')-thymidine,(0.87 g, 89%) as a mixture of E/Z isomers. The two geometrical isomerswere separated by reverse phase HPLC (Supelcostl LC18, 5μ, H₂ O: CH₃ CNgradient). (Z-isomer of title compound) ¹ H NMR (DMSO-d₆) δ 11.28 (br s,2, 2NH), 7.39 and 7.78 (2s, 2, 2C6H), 6.92 (d, 1, T1 H_(3"), J_(3'), 3",=6.7 Hz), 6.15 (pseudo t, 1, T2 H_(1'), J_(1'), 2'=7.8 Hz, J_(1'),2",=6.3 Hz), 6.04 (dd, 1, T1 H_(1'), J_(1'),2' =7.1 Hz, J_(1'),2" =6.3Hz), 5.34 (d, 1, T2 OH), 5.12 (t, 1, T1 OH), 4.11-4.25 (m, 3, T2H_(5'),5", T2 H_(3')). 3.96 (m, 1, T2 H_(4')), 3.90 (m, 1, T1 H_(4')),3.49-3.69 (m, 3, T1 H_(5'),5", T1 H_(3')), 2.06-2.31 (m,4, T1 H_(2'),2",T2 H_(2'),2") , 1.73 (s, 6, 2CH₃). Anal. Calcd. for C₂₁ H₂₇ N₅ O₉.H₂ O:C, 49.31; H, 5.72; N, 13.69. Found: C, 49.32; 5.57; N. 13.59. (E-isomerof the title compound) ¹ H NMR (DMSO-d₆) δ 11.3 (2 br s, 2, 2NH), 7.81(s, 1, C₆ H), 7.52 (d, 1, T1H_(3"), J_(3'), 3" =6.7 Hz), 7.45 (s, 1, C₆H), 6.10 (pseudo t, 1, T2 H_(1'), J_(1'),2',=7.6 Hz, J_(1'),2",=6.4 Hz),6.04 (dd, 1, T1 H_(1'J) _(1'),2' =7.3 Hz, J_(1'),2" =3.4 Hz), 5.36 (d,1, T2 OH), 5.16 (t, 1, T1 OH), 4.07-4.22 (m, 3, T2 H_(3'),5',5"), 3.91(m, 2, T1 T2 H_(4')), 3.50-3.73 (m, 2, T1 H_(5'),5"), 3.12 (m, 1, T1H_(3')), 2.05-2.44 (m, 4, T1 T2 H_(2'),2"), and 1.76 (s, 6, 2CH₃). MSFAB: M/z 494 (M+H)⁺.

EXAMPLE 10 Synthesis of phosphoramidate containing (3¹ -CH═N--O--CH₂-5') linked oligonucleoside

3'-De(oxyphosphinico)-3'-(methylidynenitrilo)-5'-O-(dimethyoxytriphenylmethyl)-thymidylyl-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The isomeric dimer of Example 9 was further dimethyoxytrityled at thehydroxyl group of the 5' terminus nucleoside followed by conversion toits 3'-O-B-cyanoethyldiisopropylphosphoramidite derivative at thehydroxyl group at the 3' terminus nucleoside of the dimer following theprocedure described in Oligonucleotide Synthesis: a practical approach,Ed. M. J. Gait, IRL Press, 1984, to yield the title compound. ¹ H NMR(CDCl₃) δ 8.77 (br s, 2, 2NH), 7.68 (s, 0.77, T1 C₆ H E-isomer), 7.59(s, 0.23, T1 C₆ H E-isomer), 6.3 (ps t, 1, T2 CH_(1')), 6.14 (m, 0.77,T1 CH_(1') E-isomer), 6.08 (m, 0.23, T₁ CH₁, Z-isomer), 1.80 and 1.50(2S, 6, 2 CH₃) and other protons. ³¹ P NMR (CDCl₃) 150.77 and 150.38(Z-isomer); 150.57 and 150.38 (E-isomer).

The protected dimer can be conveniently stored and used for couplingutilizing an automated DNA synthesizer (ABI 380B) as and when requiredfor specific incorporation into oligomers of therapeutic value. Furtheras per further examples of the specification, the oxime linked dimer isreduced to a dimer bearing a corresponding hydroxylamine linkage andthis in turn can be alkylated to a hydroxylmethylamine or otherhydroxyalkylamine linkage.

EXAMPLE 11 Synthesis of (3'-CH₂ --NH--O--CH₂ -5') linked oligonucleoside

3'-De(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(31'→5')-thymidine

To a stirred solution of blocked dimer3'-de(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidine(0.49 g, 1 mmol) in glacial AcOH (5 ml) was added NaBH₃ CN (0.19 g, 3mmol) in 3-portions under argon at room temperature. The suspension wasstirred for 1 h until bubbling of solution ceased. Additional NaBH₃ CN(0.19 g, 3 mmol) was added in a similar manner and stirring continuedfor 1 h. The AcOH was removed under reduced pressure to furnish3'-de(oxyphosphinico)-3'-(methyleneimino)-thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidine. Deblocking ofthis dimer as described before using nBu₄ NF/THF and HCl/MeOH furnishedthe title compound,3'-de(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(3'→5')-thymidine,(0.44 g, 90%) as white powder. This dimer was further purified by HPLC(as described for the3'-de(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3'→5')-thymidinedimer of Example 9) to obtain an analytically pure sample. ¹ H NMR(DMSO-d₆) δ 11.23 (br s, 2, 2NH), 7.83 and 7.49 (2s, 2, 2C₆ H), 6.82 (t,1, NHO), 6.14 (pseudo t, 1, T2 H₁, J_(1'),2',=7.6 Hz, J_(1'),2" =6.5Hz), 5.96 (dd, 1, T1 H_(1'), J_(1'),2' =6.9 Hz, J_(1'),2" =4.3 Hz), 5.28(s, 1, T2 OH), 5.08 (s, 1, T1 OH), 4.18 (m, 1, T2 H_(3')), 3.89 (m, 1,T1 H_(4')), 3.54-3.78 (m, 5, T1 T2 H_(5'),5", T2 H_(4')), 2.76-2.94 (m,2, T1 H_(3")), 2.42 (m, 1, T1 H_(3')), 2.0-2.17 (m, 4, T1, T2H_(2'),2"), 1.77 and 1.74 (2s, 6, 2 CH₃). MS FAB: M/z 496 (M+H)⁺. Anal.Calcd. for C₂₁ H₂₉ N₅ O₉.H₂ O: C, 49.12; H, 6.09; N, 13.64. Found: C,48.99; H, 5.96; N, 13.49.

EXAMPLE 12 Synthesis of methylated 3'-CH₂ --N(CH₃)--O--CH₂ -5'! linkedoligonucleoside

3'-De(oxyphosphinico)-3'- methylene(methyl-imino)!thymidylyl-(3'→5')thymidine

To a stirred solution of3'-de(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidinedimer (0.99 g, 1 mmol) in glacial AcOH (10 ml) was added aqueous HCHO(20%, 3 ml). The solution was stirred for 5 min. at room temperature andto this was added NaBH₃ CN (0.19 g, 3 mmol) in 3-portions under argon atroom temperature. The addition of NaBH₃ CN (0.19 g) was repeated oncemore and solution was further stirred for 1 h. The reaction mixture wasconcentrated to furnish crude 3'-de(oxyphosphinico)-3'-methylene(methylimino)!-thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidinedimer, which on deblocking (nBu₄ NF/THF, HCl/MeOH) furnished the titlecompound, 3'-de(oxyphosphinico)-3'-methylene-(methylimino)!thymidylyl-(3'→5') thymidine, (0.44 g, 87%) aswhite solids. The ₃ '-de(oxyphosphinico)-3'-methylene-(methylimino)!thymidylyl-(3'→5') thymidine dimer was furtherpurified by preparative HPLC furnishing an analytically pure sample. ¹ HNMR (DMSO-d₆) δ 11.30 and 11.24 (2s, 2, 2NH), 7.82 and 7.50 (2s, 2, 2C₆H), 6.15 (pseudo t, 1, T2 H_(1'), J_(1'),2' =6.3 Hz, J_(1'),2" =7.3 Hz),6.00 (pseudo t, 1, T1 H_(1'), J_(1'),2' =4.2 Hz, J_(1'),2" =6.1 Hz),5.31 (m, 1, T2 HO), 5.08 (m, 1, T1, OH), 4.17 (m, 1, T2 H_(3')), 3.88(m, 1, T2 H_(4')), 3.57-3.83 (m, 5, T₁ T2 H_(5'),5", T1 H_(4')), 2.69(m, 2, T1 H_(3")), 2.57 (s, 3, N--CH₃), 2.50 (m, 1, T1 H_(3')),2.05-2.14 (m, 4, T1 T2 H_(2'),2"), 1.79 and 1.76 (2s, 6, 2 CH₃). MS FAB:M/z 510 (M+H)⁺. Anal. Calcd. for C₂₃ H₃₁ N₅ O₉.H₂ O: C, 50.09; H, 6.31;N, 13.28. Found: C, 50.05; H, 6.21, N, 13.08.

EXAMPLE 13 Synthesis of phosphoramidate containing 3'-CH₂--N(CH₃)--O--CH₂ -5'! linked oligonucleoside

3'-De(oxyphosphinico) -3'-methylene(methylimino)!-5-O-(dimethoxytriphenylmethyl)thymidylyl-(3'.fwdarw.5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The 3'-de(oxyphosphinico)-3'- methylene(methylimino)!thymidylyl-(3'→5')thymidine dimer of Example 12 was tritylated and phosphitylated asdescribed in oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, in an overall yield of 82%. The protected dimerwas purified by silica gel column chromatography (CH₂ Cl₂ :MeOH:Et₃ N;9:1:0.1, v/v) and homogenous fractions were pooled and evaporated tofurnish pure 3'-de(oxyphosphinico)-3'-methylene(methylimino)!-thymidylyl-5'-O-(dimethoxytriphenylmethyl)-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidineas a white foam (used as such for DNA synthesis). The product wasisolated as a mixture of diastereoisomer: ³¹ P NMR (CDCl₃) δ 149.62 and149.11 ppm; ¹ H NMR (CDCl₃) δ 6.22 (pseudo t, 1, T2 H_(1'), J_(1'),2"=J_(1'),2" =6.7 Hz), 6.16 (pseudo t, 1, T1 H_(1'), J=_(1'),2' =J_(1'),2"=5.8 Hz), 2.58, 2.56 (2s, 3, N--CH₃), 1.82, 1.49 (2s, 6, 2 CH₃), andother protons.

The above protected phosphoramidate bearing dimer can be convenientlystored and used for coupling utilizing an automated DNA synthesizer (ABI380B) as and when required for specific incorporation into oligomers oftherapeutic value. Other dimers of the inventions, as for example butnot limited the above noted methylidynenitrilo, i.e., oxime, andmethyleneimino, i.e., aminohydroxy, dimers are converted to theircorresponding phosphoramidate derivatives in the same manner as thisexample and incorporated into oligonucleotide in the standard manner asnoted below. An oligomer bearing the oxime linked nucleoside dimer isreduced to an oligomer bearing a corresponding hydroxylamine linkednucleoside dimer. As noted in other examples, reduction can be effectedas an CPG bound oligomer or after removal from the CPG.

EXAMPLE 14 Synthesis of intermittent (3'-CH═N--O--CH₂ -5'), i.e., oxime;(3'-CH₂ --NH--O--CH₂ -5'), i.e., aminohydroxy; (3'-CH₂--N(CH₃)--O--CH2-5'), i.e., N-methyl-aminohydroxy; (3'-CH₂--O--N(CH₃)--CH₂ -5'), i.e., N-methyl-hydroxyamino; or (3'-CH₂--N(CH3)--N(CH₃)--CH₂ -5'), i.e., N,N'-dimethyl-hydrazino linkedoligonucleosides

An appropriate 2'-deoxynucleoside that will become the 3'-terminalnucleoside of an oligonucleoside is bound to a CPG column for use on anABI 380B automated DNA synthesizer. Standard phosphoramidite chemistryprogram steps were employed to place the dimer bearing the(3'-CH═N--O--CH₂ --5'), i.e., oxime; (3'-CH₂ -NH-O-CH₂ -5'), i.e.,aminohydroxy; (3'-CH₂ --N(CH₃)--O--CH₂ -5'), i.e.,N-methyl-aminohydroxy; (3'-CH₂ --O--N(CH₃)--CH₂ -5'), i.e.,N-methyl-hydroxyamino; or (3'-CH₂ --N(CH₃)--N(CH₃)-CH₂ -5'), i.e.,N,N'-dimethylhydrazino, linkages into the desired position or positionsof choice within the sequence.

EXAMPLE 15 General and specific NaBH₃ CN reduction for conversion of(3'-CH═N--O--CH₂ -5') linkage to (3'-CH₂ --NH--O--CH₂ -5') Reduction ofa Dimer

To a solution of a dimer (0.49 g, 1 mmol) in glacial acetic acid (AcOH)(5 ml) was added sodium cyanoborohydride (0.19, 3 mmol) in ACOH (1 ml),under an argon atmosphere at room temperature. The suspension wasstirred for 1 h, and an additional amount of NaBH₃ CN in ACOH (1 ml) wasadded and stirring continued for 1 h. The excess of AcOH was removedunder reduced pressure at room temperature. The residue was coevaporatedwith toluene (2×50 ml) and purified by silica gel (25 g) columnchromatography. Elution with CH₂ Cl₂ :MeOH (9:1, v/v) and pooling ofappropriate fractions, followed by evaporation furnished 0.36 g (75%) ofsolid dimer.

Reduction of an Oligonucleoside

CPG-bound oligonucleoside (1 MM), that contains one (or more) backbonemodified linkages is taken off the DNA synthesizer after completion ofits synthesis cycles. A 1.0 M NaBH₃ CN solution in THF:AcOH (10 ml, 1:1v/v) is pumped through the CPG-bound material in a standard wayutilizing a syringe technique for 30 min. The column is washed with THF(50 ml), and the reduced oligonucleoside is released from the supportcolumn in a standard way.

Alternative Reduction of an oligonucleoside

As an alternative to the above reduction, reduction can also beaccomplished after removal from the CPG support. At the completion ofsynthesis the oligonucleoside is removed from the CPG-support bystandard procedures. The 5'-O-trityl-on oligonucleoside is purified byHPLC and then reduced by the NaBH₃ CN/AcOH/THF method as describedabove.

EXAMPLE 16 Synthesis of (3'-CH₂ --N(CH₃)--O--CH₂ -5') linkedoligonucleoside having a 2',3'-didehydro nucleoside as its 5' terminalnucleoside

3'-De(oxyphosphinico)-2', 3-didehydro-3'-methylene(methylimino)!thymidylyl-(3'→5')thymidine.

To a stirred solution of1-(5'-O-(MMTr)-β-D-glycero-pentofuran-3'-ulosyl!thymine (0.13 mmol;prepared according to the procedure of T.-C. Wu, et al., Tetrahedron,1989, 45:855, 5'-O-(methyleneamino)-3'-O-(t-butyldiphenylsilyl)thymidine(0.13 mmol; prepared according to the procedure of Debart, et al.,Tetrahedron Letters 1992, 33, 2645, ethylene glycol (0.5 mmol), and HMPA(0.5 ml) was added SmI₂ in THF (0.1 mol, 3 ml, 3 mmol) at roomtemperature. The color of SmI₂ fades out as the reaction proceeds toform the desired adduct. After complete disappearance of startingmaterials the reaction mixture is worked-up in the usual way. (Theproduct could be purified by silica column chromatography forcharacterization). The crude mixture of 3'-epimeric adduct is thenalkylated (HCHO/NaCNBH₃ /ACOH) as described in other of these examples.The methylated product is then treated with methylsulfonylchloride inpyridine to obtain a 3'-epimeric mesylate, which on base treatment wouldfurnish the title compound.

EXAMPLE 17 Synthesis of (3'-CH2--CH2--NH--CH₂ -5') linkedoligonucleoside

3'-De(oxyphosphinico)-3'-(1,2-ethanediylimino)-thymidylyl-5'-O-(t-butyldimethylsilyl)-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine

To a stirred solution of aldehyde 2.5 g, 6.5 mmol, freshly preparedaccording to the procedure described by Fiandor, et al., TetrahedronLetts. 1990, 33, 597!,5'-amino-3'-0-(t-butyldiphenylsilyl)-5'-deoxythymidine 3.13 g, 6.5 mmol,prepared in two steps via 3'-O-silylation of 5'-azido-5'-deoxythymidinein the manner of Hata, et al., J. Chem. Soc. Perkin I 1980, 306, andsubsequently reduction of the product by the method of Poopeiko, et al.,Syn. Lett. 1991, 342!, AcOH (0.39, and 6.5 mmol) in dicholoroethane (65ml) was added followed by NaBH(OAc)₃ (2.759, 13.08 mmol) under argon.The suspension was stirred for 3 hours at room temperature. The reactionmixture was diluted with CH₂ Cl₂ (250 ml) and washed with water (2×100ml). The organic layer was dried (MgSO₄) and concentrated to furnish thecrude product as a syrup. The product was purified by silica gel columnchromatography to furnish the title compound as white foam (3.5 g, 64%).¹ H NMR (CDCl₃) δ 0.1 s, 6, Si(CH₃)₂ !; 0.9 and 1.1 2s, 18, 2 Si(CH₃)₃!1; 1.85 and 1.95 (2s, 6, 2 CH₃); 2.5 (m, 2, 3"CH₂); 3.7 (m, 2, 5¹ CH2);4.0 (m, 2, 31,41 CH); 4.2 (m, 1, 3'CH); 6.05 (m, 1, 1'H); 6.28 (t, 1,1'H); 7.1 and 7.57 (2s, 2, C6H); 7.35-7.7 2m, 12, Si ArH)₂ !, and othersugar protons.

3'-De(oxyphosphinico)-3'-(1,2-ethanediylimino)-thymidylyl-(3'→5')-5'-deoxythymidine

The protected dimer was deblocked in 81% yield following the standardprocedure using (Bu)₄ NF in THF. The deblocked dimer was purified byHPLC for analysis. ¹ H NMR (DMSO-d₆) δ 1.76 and 1.78 (2s, 6, CH₃);2.0-2.2 (3m, 4,2'CH₂); 3.15 (m, 2, NCH₂); 3.56 (m, 2, 4'H, 5'CH₂); 4.18(br s, 1, 3'H); 5.17 and 5.22 (2 br s, 2, 2 OH); 5.95 (t, 1,1'H); 6.1(t, 1, 1'H); 7.6 and 7.85 (2s, 2, 2(C₆ H)); 11.25 (br s, 2 2NH) andother protons.

EXAMPLE 18 Synthesis of Monomer Unit for (3'-CH;--O--N═CH-5'), (3'-CH₂--O--NH--CH₂ -5') and (3'-CH₂ --O--N(CH₃)--CH₂ -5') Linkages

1-3'-Deoxy-3'-C-(hydroxymethyl)-5'-O-(trityl)-β-D-erythro-pentofuranosyl!-thymine

A suspension of NaBH₄ (1.36 g, 9.6 mmol) was added dropwise to a stirredsolution of 3'-C-formyl-5'-O-trityl-thymidine in EtOH:H₂ O (22 ml, 3:1,v/v) mixture at room temperature. After 3 h, EtOAc (300 ml) was addedand the organic layer was washed with H₂ O (2×150 ml). The dried (MgSO₄)EtOAc extract was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(9:1, v/v), pooling and concentration of appropriate fractions gave thetitle compound (1.13 g, 83%). ¹ H-NMR (CDCl₃) δ 8.29 (br s, 1, NH), 7.59(s, 1, C₆ H) 7.47-7.22 (m, 15, TrH) 6.13 (dd, 1, H₁ ', J_(1'),2' =6.5Hz); 3.98 (m, 1, H₄ ); 3.62 (m, 2, H₃ "), 3.56-3.33 (m, 2, H₅ ',H₅ "),2.60 (m, 1, H₃ '); 2.33-2.20 (m, 2, H₂ ' H₂ '); 1.91 (br s, 1, OH); 1.53(S, 3, CH₃).

1- 3'-Deoxy-3'-C-O-(phthalimidohydroxymethyl)!-5'-O-trityl-β-D-erythro-pentofuranosyl!-thymine

Diisopropylazodicarboxylate (0.47 ml, 2.41 mmol) was added to a stirredsolution of 3'-deoxy-3'-C-(hydroxy-methyl)-5'-O-trityl-thymidine (0.8 g,1.62 mmol), N-hy-droxyphthalimide (0.35 g, 2.15 mmol),triphenylphosphine (0.56 g, 2.15 mmol) in dry THF (10 ml) at roomtemperature. After 48 h, the products were concentrated and the residuewas extracted with CH₂ Cl₂ (2×100 ml). The CH₂ Cl₂ extracts were washedwith NaHCO₃ (5%, 100 ml) and water (100 ml). The dried (MgSO₄) extractwas evaporated under reduced pressure and the residue was purified byshort-silica gel chromatography. Elution with EtOAC:Hexanes (1:1, v/v),pooling and concentration of appropriate fractions gave the titlecompound as white foam (0.82 g, 79%). ¹ H-NMR (CDCl₃) δ 8.24 (s, 1, NH);7.85-7.20 (m, 20, TrH, ArH, C₆ H), 6.20 (m, 1, H₁,), 4.22-4.16 (m, 3, H₄', H₃ "), 3.63-3.40 (m, 2, H_(5'),H_(5")), 3.02 (m, 1, H_(3')),2.50-2.43 (m, 2, H₂, H_(2")); 1.51 (s, 3, CH₃). Anal. Calcd. for C₃₈ H₃₃N₃ O₇. 0.5 EtOAc:C, 69.86; H, 5.42, N, 6.11. Found: C, 70.19; H, 5.27;N, 5.75

1-{3'-Deoxy-3'-C- O-(aminohydroxymethyl)!-5'-O-15trityl-β-D-erythro-pentofuranosyl}-thymine

Methylhydrazine (0.12 ml, 2.25 mmol) was added to a stirred solution of3'-deoxy-3'-C- O-(phthalimidohydroxymethyl)!-5'-O-tritylthymidine (0.77g, 1.2 mmol) in dry CH₂ Cl₂ (9 ml) at room temperature. After 1 h, theprecipitate was filtered and the residue washed with CH₂ Cl₂ (2×10 ml).The combined filtrates were concentrated and the residue was purified bysilica gel column chromatography. Elution with CH₂ Cl₂ :MeOH (97:3,v/v), pooling and evaporation of appropriate fractions gave the titlecompound as white powder (0.43 g, 70%). ¹ H-NMR (CDCl₃) δ 8.59 (br s, 1,NH), 7.66 (m, 1, C₆ H), 7.40-7.15 (m, 15, TrH), 6.06 (pseudo t, 1, H1,),5.22 (br s, 2, NH₂), 3.89 (m, 1, H_(4')), 3.65-3.20 (m, 4, H_(5'),H_(5"), H_(3")), 2.81 (m, 1, H_(3')), 2.21-2.13 (m, 2, H_(2'), H_(2")),1.37 (s, 3, CH₃).

EXAMPLE 19 Synthesis of (3'-CH₂ --O--N═CH-5'), (3'-CH₂ --O--NH--CH₂ -5')and (3'-CH₂ --O--N(CH₃)-CH₂ -5') linked oligonucleosides

3'-De(oxyphosphinico)-3'- methyleneoxy(methylimino)!thymidylyl-(3'→5')-5'-deoxythymidine

A mixture of 1-4-C-formyl-3-O-(t-butyldiphenylsilyl)-β-D-erythro-pentofuranosyl)thymine1 mmol, prepared according to the procedure of Nucleosides andNucleotides 1990, 9, 533!, 3'-deoxy-3'-C-(O-(aminohydroxymethyl)!-5'-0-tritylthymidine (1 mmol), ACOH (0.1 ml),and dry CH₂ Cl₂ (25 ml) was stirred at room temperature for 1 h. Thesolvent was evaporated and the residue was dissolved in glacial AcOH (5ml). NaBH₃ CN (3 mmol) was added to the stirred AcOH reaction mixture.After 1 h, an additional amount of NaBH₃ CN (3 mmol) was added and themixture stirred for 1 h. The reaction was concentrated under vacuum andthe residue was purified by silica gel column chromatography to furnish5'-O-Tr-T-3'-CH₂ --O--NH--CH₂ -5'-T-3'-O-TBDPSi dimer. ¹ H-NMR (CDCl₃) δ8.73 (br s, 2, 2NH), 7.67 (s, 1 C₆ H), 7.674-7.23 (m, 20, TrH, TBDPhH),6.96 (s, 1, C₆ H), 6.23 (pseudo t, 1, T₂ H_(1')), 6.11 (pseudo t, 1, T₁,H_(1')) 5.51 (br s, 1, NH), 4.16 (m, 1, T₂ H_(3')) 4.02 (m, 1, T₂H_(4')), 3.87 (m, 1, T₁ H_(4')), 3.52 (m, 3, T₁ CH_(23"), T₁ H_(5")),3.23 (m, 1, T₁ H₅ '), 2.55-2.76 (m, 3, T1 H_(3'), T2 H_(5') H_(5")),2.33-2.27 (m, 1, T2 H_(2')), 2.23-2.12 (m, 2, T1 H₂ H_(2")), 1.95-1.85(m, 1, T₂ H_(2")), 1.83 (s, 3, CH₃) 1.45 (s, 3, CH₃), 1.06 (s, 9, (CH₃)₃CSi).

The latter dimer was methylated using HCHO/NaBH₃ CN/AcOH and finallydeblocked with nBu₄ NF/THF and HF/CH₃ CN in two-steps to furnish thetitle compound (65% yield). ¹ H-NMR (DMSO-d₆) δ 11.27 (br s, 2, NH);7.85 (s, 1, T1 C₆ H); 7.51 (s, 1, T₂ C₆ H); 6.15 (pseudo t, 1, T₂ H₁,J_(1'-2') =7.8 Hz, J_(1'-2") =6.3 Hz); 6.00 (pseudo t, 1, T₁ H_(1'),J_(1'-2') =6.9 Hz, J_(1'-2") =4.5 Hz), 5.32 (br s, 1, OH_(3')), 5.09 (brs, 1, OH5'); 4.17 (m, 1, T₂ H_(3')); 3.90 (m, 1, T₂ H₄); 3.76-3.66 (m,4, T₁ H_(4'), T₁ H_(5'), CH₂ 3"); 3.60-3.52 (m, 1, T₁ H_(5")); 2.82 (m,2, T₂ H_(5'), H_(5")); 2.57 (s, 3, N--CH₃); 2.47 (m, 1, T₁ H_(3'));2.23-2.02 (m, 4, H_(2') H_(2")) 1.81 (s, 3, C₅ CH₃); 1.78 (s, 3, C₅CH₃). Anal. Calcd. for C₂₂ H₃₁ N₅ O₉.O.5 H₂ O: C, 50.96; H, 6.22; N,13.50. Found: C, 51.01; H, 6.22; N, 13.19. MS (FAB+, glycerol) M+H⁺ m/z=510.

EXAMPLE 20 Synthesis of phosphoramidate containing (3'-CH₂--O--N(CH₃)--CH₂ -5') linked oligonucleoside

3'-De(oxyphosphinico)-3'-methyleneoxy(methylimino)!-thymidylyl-5'-O-(dimethyoxytriphenylmethyl)-(3'→5')-3'-(O-β-cyanoethyldiisopropylaminophosphiryl)thymidine

Dimethyoxytritylation of the dimer 5'-OH-T-3'-CH₂ --O--NCH₃ --CH₂-5'-T-3'-OH following the procedure described in OligonucleotideSynthesis: a practical approach, Ed. M. J. Gait, IRL Press, 1984,furnished the 5'-O-DMTr protected dimer as white foam. ¹ H-NMR (CDCl₃) δ7.67 (s, 1, H₆); 7.44-6.82 (m, 14, H₆, DMTrH); 6.20 (pseudo t, 2,H_(1')); 4.3 (m, 1, T₂ H_(3')); 4.15 (m, 1, T₂ H_(4')); 4.00 (m, 1, T₁H_(4')); 3.80 (s, 6, OCH₃); 3.77-3.23 (m, 4, T₁ H_(5') H_(5"), CH₂ 3");2.89-2.50 (m, 3, T₂ H_(5') H_(5"), T1 H₃ '); 2.62 (s, 3, N--CH₃);2.48-2.08 (m, 4, H_(2') H_(2")); 1.9 (s, 3, C₅ CH₃) 1.48 (s, 3 C₅ CH₃).

Above compound was phosphitylated following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, to furnish the title compound in 70% yield over two stepsas white powder. ¹ H NMR (CDC1₃) δ 8.25 (br s, 2, NH), 7.66 (s, 1, C₆H), 7.15-7.45 (m, 10, ArH, C₆ H), 6.8-6.9 (m, 4, ArH), 6.12 (m, 2,2C_(1') H), 3.79 (s, 6, ArOCH₃), 2.56 (s, 3, N--CH₃), 1.88, 1.44 (2s, 6,2 C₅ CH₃) and other protons. ³¹ P NMR (CDCl₃) 149.42 and 148.75 ppm.

EXAMPLE 21 Synthesis of oligonucleosides having linkages that includepharmacokinetic and pharmacodynamic property modifying groups locatedtherein on

3'-De(oxyphosphinico)-31-methylene(benzylimino)!-thymidylyl-5'-O-(dimethyoxytriphenylmethyl)-(31'→5')-3'-O-β-(cyanoethyldiisopropylaminophosphiryl)thymidine

A reductive coupling of 3'-deoxy-3'-C-formyl-5'-O-tritylthymidine (1.5mmol) with 5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine (1.5 mmol) asdescribed in Example 9 furnished 5'-O-Tr-T-3'-CH₂ --NH--O--CH₂-5'-T-3'-O-TBDPSi dimer. This dimer was benzylated with C₆ H₅ CHO/NaBH₃CN/AcOH in the same manner as the above described methylation to yieldN-benzylated dimer 5'-O-Tr-T-3'-CH₂ --NBz--O--CH₂ -5'-T-3'-O-TBDPSi. Thelatter dimer was deblocked using nBu₄ NF/THF and HCl/MeOH methodology asdescribed in above examples to furnish deblocked dimer 5'-OH-T-3'-CH₂--NBn--O--CH₂ -5'-T-3'-OH, which on dimethoxytritylation and subsequentphosphitylation following the procedure described in OligonucleotideSynthesis: a practical approach, Ed. M. J. Gait, IRL Press, 1984, gavethe title compound (45% overall yield). ¹ H NMR (CDCl₃) δ 6.15 (pseudot, 1, T2 C_(1') H); 6.09 (m, 1, T1 C_(1') H); 3.76 (s, 6, 2OCH₃); 1.7and 1.48 (2S, 6, 2-CH₃) and other protons. ³¹ p NMR (CDCl₃) 149.59 and149.23 ppm.

The phosphiltylated dimer was successfully incorporated into an oligomerusing an automated DNA synthesizer in the manner of Example 8illustrating the ability to attach of various pharmacokinetic andpharmacodynamic property modifying groups into the backbone linkageprior to the DNA synthesis of an oligonucleotide.

EXAMPLE 22 Synthesis of (3'--CH₂ --NH--CH₂ --CH₂ -5'), (3-CH₂--N(CH₃)--CH₂ -CH₂ --5'), and Phosphoramidate Derivative

3'-De(oxyphosphinico-3'-(methyleneimino)-methylene!-5'-O-(dimethyoxytrityl)thymidylyl-(3'→5')-thymidine

A reductive amination according to the procedure of Abdel-Magid, et al.,Tetrahedron Letts. 1990, 31, 5595! of3'-deoxy-3'-C-formyl-5'-O-tritylthymidine (1 mmol) with 1- 6'-amino-2',5',6'-trideoxy-3'-O-(t-butyldiphenylsilyl)-β-D-erythro-hexofuranosyl!thymine1.2 mmol, prepared according to the procedure of Etzold. et al., J. C.S. Chem. Comm. 1968, 422! in presence of ACOH gave a blocked dimer5'-O-Tr-T-3'-CH₂ NH--CH₂ --CH₂ -5'-T-3'-O-TBDPSi, which on deprotectionas described in above examples gave 5'-OH-T-3'-CH₂ --NH--CH₂ --CH₂-5'-T-3'-OH dimer as white powder (70% yield). ¹ H NMR (D₂ O, pH 5.6,20° C.) δ T1thymidine unit: 7.78 (s, 1, C₆ H); 6.17 (t, 1, C₁ H); 4.45(m, 1, C_(3') H); 4.08 (m, 1, C_(4') H); 4.00, 3.72 (m, 2, C_(5'),5" H);2.9 (m, 2 C_(6'),6" H); 2.34 (m, 2, C_(2'),2 H); 1.77 (s, 3, CH₃); T2thymidine unit: 7.47 (s, 1 C₆ H); 6.07 (t, 1, C_(1') H); 3.89 (m, 2,C_(5'5") H); 3.79 (m, 11 C_(4") H); 2.89 (m, 1, C_(3") H); 2.38 (m, 1,C_(2') H); 2.32 (m, 1, C_(3') H); 1.72 (s, 3, CH₃); and 2.68 (s,N--CH₃).

Pka determination:

The sensitivity of the proton chemical shift of the N-Me group of theforegoing dimer to change in response to change in pH was measured byNMR as an indicator of the pka of the backbone amine. The chemical shiftmoved downfield as the amino group was protonated. A 4 mg sample of5'-OH-T-3'-CH₂ --NCH₃ --CH₂ --CH₂ -5'-T-3'-OH dimer was dissolved in 0.6ml of 30 mM bicarbonate buffer. The pH was varied between 5.1 and 10.0using 0.1 N NaOH in 6-steps. The chemical shift of the N-methyl protonvaried between 2.26 and 2.93 ppm, giving rise to a pka of 7.8±0.1. Whilewe do not wish to be bound by theory, it is thus believed that atphysiological pH this backbone will be protonated.

3'-De(oxyphosphinico-3'-methylene(methylimino)-methylene!-5'-O-(dimethyoxytrityl)-thymidylyl-(31'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The proceeding dimer was methylated using HCHO/NaBH₃ CN in ACOH tofurnish 5'-OH-T-3'-CH₂ --N(CH₃)--CH₂ --CH₂ -5'-T-3'-OH dimer, which ondimethoxytritylation and phosphitylation following the proceduredescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, gave the title compound as foam (68% yield). ¹ HNMR (CDCl₃) δ 6.12 (m, 2, 2C_(1') H); 2.15, 2.14 (2s, 3, N--CH₃); 1.88,1.45 (2s, 6, 2 C₅ CH₃) and other protons. ³¹ P NMR (CDC1₃) 149.49 and148.96 ppm.

EXAMPLE 23 (3'-CH₂ --N(labile blocking group)-O-CH₂ -5') dimer andphosphoramidate derivative--a dimer Incorporating a3'-de(oxyphosphinico)-3'-(methyleneimino) (3→5') linkage having a labileN-protecting group for regeneration of a (3'-CH₂ -NH-O-CH₂ -5) linkage

3'-DeCoxyphosphinico)-3'-methylene(phenoxyacetylimino)!-thymidylyl-(3'.fwdarw.5')-thymidine

To a stirred solution of 5'-O-Tr-T-3'-CH₂ --NH--O--CH₂ -5'-T-3'-O-TBDPSi(1 mmol, prepared according to the procedure of Debart, et al.,Tetrahedron Letts. 1992, 33, 2645) in dry pyridine (10 ml) was addedphenoxyacetylchloride (1.2 mmol). After 12 h, the products were dilutedwith CH₂ Cl₂ (200 ml) and washed with sat. NaHCO₃ (2×50 ml), water (2×50ml) and dried (MgSO₄). The CH₂ Cl₂ extract was concentrated and residuepurified by silica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(9:1, v/v), pooling of appropriate fractions and evaporation furnished5'-O-Tr-T-31-CH₂ --N(COCH₂ OPh)-O-CH₂ -5'-T-3'-O-TBDPSi dimer as whitefoam. ¹ H NMR (DMSO-d₆) δ 11.35 (br s, 2, NH); 7.6-6.65 (m, 32, Tr,TBDPS, phenoxyacetyl, C₆ H); 6.3 (pseudo t, 1, H_(1')); 6.03 (pseudo t,1, H_(1')); 4.5 (m, 2, CH₂); 4.3 (m, 1, T₂ H₃); 3.9-3.3 (m, 6, T₁H_(4'), T₂ H_(4'), T₂ H_(4'), T₂ H_(5') H_(5"), CH_(23")); 3.10 (m, 2,T, H_(5') H_(5")); 2.65 (m, 1, T₁ H_(3')); 2.2-2.05 (m, 4, H_(2')H_(2")); 1.58 (s, 3, CH₃); 1.4 (s, 3, CH₃); 1.02 (s, 9, (CH₃)₃ CSi).

The foregoing dimer was sequentially deblocked with HF (48%)/CH₃ CN(5:95, v/v) treatment to remove the trityl group, and the product ontreatment with nBu₄ NF/THF removed the silyl group to furnish titlecompound as white powder (70% yield for 3-steps). ¹ H NMR (DMSO-d₆) δ11.35 (br s, 1, NH); 11.25 (br s, 1, NH) 7.92 (s, 1, C₆ H); 7.5 (s, 1,C₆ H); 7.2-6.8 (m, 5, ArH); 6.23 (pseudo t, 1, H_(1')); 5.98 (dd, 1,H_(1')); 5.45 (d, 1, OH_(3')), 5.15 (t, 1, OH_(5')); 4.9 (m, 2, CH₂);4.3-3.5 (m, 9, T₂ H_(3'), H_(4'), H_(5') H_(5"), CH_(23")); 2.6 (m, 1,T₁ H_(3')); 2.25-2.00 (m, 4, H_(2') H_(2")); 1.75 (s, 3, CH₃); 1.65 (s,3, CH₂).

The latter dimer was dimethoxytritylated as per the procedure ofdescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, 1984, IRL Press, to furnish 5'-O-DMT-T-3'-CH₂ -N-(COCH₂ OPh)-O-CH₂-5'-T-3'-OH as pale yellow colored foam. ¹ H NMR (DMSO d₆) δ 11.3 (br s,2, NH); 7.55 (s, 1, C₆ H). 7.45 (s, 1, C₆ H); 7.38-6.75 (m, 18, DMTrH,phenoxyacetyl-H); 6.22 (pseudo t, 1, T₂ H_(1')); 6.05 (pseudo t, 1, T₁H_(1')); 4.75-4.60 (m, 2, CH₂); 4.25 (m, 1, T₂ H_(5')); 4.18 (m, 1, T₂H_(3')); 4.05 (m, 1, T₂ H_(5")); 3.9 (m, 2, H_(4')); 3.8-3.6 (m, 2,CH_(23")); 3.65 (s, 6, 2OCH₃) 3.2 (m, 2, T₁, H_(5') H_(5")) 2.82 (m, 1,T₁ H_(3')); 2.3-2.05 (m, 4, H_(2') H_(2")); 1.6 (s, 3, T₂ CHCH₃); 1.38(s, 3, T1 CH₃).

The above dimer on phosphitylation following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, 1984,IRL Press, furnished the phosphoramidate derivatized dimer (appropriatefor use on DNA synthesizer) as a foam (75% in 2 steps). ¹ H NMR (CDCl₃)δ 7.62 (s, 1, C₆ H); 7.2-7.45 (2m, 12, ArH); 6.77-7.05 (3m, 7, ArH, C₆H); 6.15 (pseudo t, 1, C_(1') H); 6.05 (t, 1, C_(1') H); 4.7 (m, 2,2C_(4') H); 3.74 (2s, 6, 2ArOCH₃); 2.95 (m, 1, C_(3") H); 1.78, 1.77(2s, 3, C₅ CH₃); 1.41 (s, 3, C₅ CH₃), and other protons. ³¹ P NMR(CDCl₃) 1.49.76 and 149.56 ppm.

EXAMPLE 24 Regeneration of (3'-CH₂ -NH--O--CH₂ -5') linkage from (3'-CH₂--N(labile blocking group)--CH₂ --CH₂ -5') linkage In an oligonucleotide

The phosphitylated dimer of Example 23 will be incorporated within anoligonucleotide as per the procedure of Example 8. After completion ofthe oligonucleotide on the support, the oligonucleotide is cleaved fromthe support utilizing standard ammonium hydroxide conditions. Concurrentwith the cleavage from the support the ammonium hydroxide treatment willfurther cleave the phenoxyacetyl blocking group from the imino nitrogenof the incorporated (3'-CH₂ --N(COCH₂ OPh)--O--CH₂ -5') oligonucleosidedimer to yield the (3'-CH₂ --NH--O--CH₂ -5') linked oligonucleosidedimer within the oligonucleotide structure.

EXAMPLE 25 5'-O-(t-Butyldimethylsilyl)-3'-O-Phthalimidothymidine, 2

To a solution of 5'-O-t-butyldimethylsilylthymidine 1, 21.36 g, 60 mmol,prepared according to the procedure of Nair, et al., Org. Prep.Procedures Int. 1990, 22, 57 in dry THF (750 ml)!, triphenylphosphine(17.28 g, 66 mmol) and N-hydroxyphthalimide (10.74 g, 66 mmol) wereadded. The solution was cooled to 0° C. and diisopropylazodicarboxylate(15.15 g, 75 mmol) was added dropwise over a period of 3 hr whilestirring under nitrogen. The reaction mixture was then stirred at roomtemperature for 12 hr. The solution was evaporated and the residue wasdissolved in CH₂ Cl₂ (750 ml), extracted with sat. NaHCO₃ (200 ml), andwater (200 ml), dried (MgSO₄), filtered and concentrated to furnishyellow oily residue. Silica gel column chromatography (100% hexanes, andthen hexanes:Et₂ O gradient to 90% Et₂ O) of the residue gave compound 2as a colorless glass (18.68 g, 62%); ¹ H NMR (CDCl₃) δ 0.05 2s, 6,(CH₃)₂ !, 0.91 s, 9, (CH₃)₃ !, 2.0 (s, 3, CH₃), 2.5-2.65 (m, 2, 2'CH₂),4.05-4.2 (m, 2, 5'CH₂), 4.25-4.35 (m, 1, 4'H), 5.0 (m, 1, 3'H), 6.15 (m,1'H), 8.6 (br s, 1, NH), and aromatic protons. Anal. Calcd. for C₂₄ H₃N₃ O₇ Si: C, 57.46;H, 6.23; N, 8.37. found : C, 57.20; H, 6.26; N, 8.27.

EXAMPLE 26 3'-O-Amino-5'-O-(t-Butyldimethylsilyl)thymidine, 3

Cold methylhydrazine (1.6 ml, 30 mmol) was added to a stirred solutionof 5'-O-(t-butyldimethylsilyl)-3'-O-phthalimidothymidine (2, 4.6 g, 9.18mmol) in dry CH₂ Cl₂ (60 ml) at 5°-10° C. After 10 minutes whiteprecipitation of 1,2-dihydro-4-hydroxy-2-methyl-1-oxophthalizineoccurred. The suspension was stirred at room temperature for 1 h. Thesuspension was filtered and precipitate washed with CH₂ Cl₂ (2×20 ml).The combined filtrates were concentrated and the residue purified bysilica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(100:0→97:3, v/v) furnished the title compound (3.40 g, 100%) as whitesolid. Crystallization from CH₂ Cl₂ gave white needles, m.p. 171° C.; ¹H NMR (CDCl₃) δ 0.05 s, 6, (CH₃)₂ !, 0.90 s, 9, (CH₃)₃ !, 2.22-2.58 (2m,2, 2'CH₂), 3.9-4.08 (m, 3, 5'CH₂, and 3'H) 4.30 (m, 1, 4'H) 5.5 (br s,2, NH₂) 6.2 (m, 1, 1'H) 7.45 (s, 1, C₆ H) 8.9 (br s, 1, NH). Anal.Calcd. for C₁₆ H₂₉ N₃ O₅ Si: C, 51.72; H, 7.87; N, 11.32. found: C,51.87, H, 7.81; N, 11.32.

EXAMPLE 27 3'-O-Aminothymidine, 4

3'-O-Amino-(t-butyldimethylsilyl)thymidine was deblocked with (Bu)₄NF/THF in standard way to furnish compound 4 (72%). Crystallized fromether/hexanes/ethanol as fine needles, mp 81° C. ¹ H NMR (Me₂ SO-d₆) δ1.78 (s, 3, CH₃), 2.17 and 2.45 (2m, 2, 2'CH₂), 3.70 (m, 2, 5'CH₂), 3.88(m, 1, 4'H), 4.16 (m, 1, 3'H), 4.8 (br s, 1, 5'OH), 6.05 (dd, 1, 1'H),6.2 (br s, 2 NH₂), 7.48 (s, 1, C₆ H), and 11.24 (br s, 1NH). Anal.Calcd. for C₁₀ H₁₅ N₃ O₅ : C, 46.69; H, 5.87; N, 16.33; found: C, 46.55;H, 5.91; N, 16.21.

EXAMPLE 283'-O-Dephosphinico-3'-O-(Methylimino)thymidylyl-(3'→5')-5'-Deoxythymidine,9

Step 1.

3'-O-Amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 1.85 g, 5 mmol),3'-O-(t-butyldimethylsilyl)thymidine-5'-aldehyde 5, 2.39 g, 5 mmol;freshly prepared by following the method of Camarasa, et al.,Nucleosides and nucleotides 1990, 9, 533! and AcOH (0.25 ml) werestirred together in CH₂ Cl₂ (50 ml) solution at room temperature for 2h. The products were then concentrated under reduced pressure to givethe intermediate oxime linked dimer, compound 6.

Step 2.

The residue obtained from Step 1 was dissolved in AcOH (25 ml). NaCNBH₃(1.55 g, 25 mmol, in 3-portions) was added to the stirred ACOH solutionat room temperature. The solution was stirred for 30 min to give theintermediate imine linked dimer, compound 7.

Step 3.

Aqueous HCHO (20%, 2 ml, 66 mmol) and additional NaCNBH₃ (1.55 g, 25mmol, in 3-portions) was added to the stirred reaction mixture of Step 2at room temperature. After 2 h, the solution was diluted with EtOH (100ml), and resulting suspension was evaporated under reduced pressure. Theresidue was dissolved in CH₂ Cl₂ (150 ml) and then washed successivelywith 0.1M HCl (100 ml), saturated aqueous NaHCO₃ (100 ml), and water(2×50 ml). The dried (MgSO₄) CH₂ Cl₂ solution was evaporated to givecrude methylated imine linked dimer 8.

Step 4.

The residue from Step 3 was dissolved in the THF (30 ml) and a solutionof (Bu)₄ NF (1M in THF, 10 ml) was added while stirring at roomtemperature. After 1 h, the reaction mixture was evaporated underreduced pressure and the residue was purified by short columnchromatography. The appropriate fractions, which eluted with CH₂ Cl₂:MeOH (8:2, v/v) were pooled and evaporated to give compound 9 as a foam(0.74 g, 30%). ¹ H NMR (Me₂ SO-d₆) δ 1.78 (s, 6, 2CH₃), 2.10 (m, 4,2'CH₂), 2.5 (s, 3, N--CH₃), 2.8 (m, 2, 5'-N--CH₂), 3.6-4.08 (5 m, 6,5'CH₂, 4'CH, 3'CH), 4.75 and 5.3 (2 br s, 2, 3' and 5' OH), 6.02 (d, 1,1'H), 6.1 (t, 1, 1'H), 7.4 and 7.45 (2s, 2, 2C₆ H), 11.3 (br s, 2, NH).

EXAMPLE 29 Methyl3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-5-C-Formyl-α/β-D-erythro-Pentofuranoside,23

2-Deoxy-D-ribose, 21, was modified to methyl2-deoxy-α/β-D-erythro-pentofuranoside (prepared according to the methodof Motawai, et al., Liebigs Ann. Chem. 1990, 599-602), which onselective tosylation followed by 3-O-silylation gave methyl3-O-(t-butyldimethylsilyl)-2-deoxy-5-O-tosyl-α/β-D-erythro-pentofuranosidein overall 70% yield. The latter compound on iodination followed bycyanation gave the corresponding 5-C-cyano intermediate compound 22, asa syrup. ¹ H NMR (CDCl₃) δ 1.05 (s, 9, (CH₃)₃), 1.9-2.38 (m, 4, 2 CH₂),3.3 and 3.4 (2s, 3, OCH₃), 3.98-4.30 (3m, 2, 3, 4-CH), 4.95 and 5.05 (2m, 1, 1H), 7.4 and 7.7 (2m, 10, Ph H). IR (neat) 2253 cm⁻¹ (CH₂ CN)!.Compound 22 (stored at 0° C. without any degradation) wasreduced(DIBAL-H) freshly every time as and when the title compound 23was required.

EXAMPLE 30 5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'(Methyleneamino)oxyladenosine, 27;5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-(Cethyleneamino)oxy!cytidine, 28; and5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-(Methylene-amino)oxy!guanosine, 29

3'-O-Amino-2'-deoxyadenosine, compound 24, 3'-O-amino-2'-deoxycytidine,compound 25, and 3'-O-amino-2'-deoxyguanosine, compound 26, prepared asper the procedures of European Patent Application 0 381 335 or in amanner analogous to the preparation of compound 4 by the procedure ofExample 27 above, are blocked at their 5' position with at-butyldimethylsilyl group according to the procedure of Nair, et al.,Org. Prep. Procedures Int. 1990, 22, 57, to give the corresponding3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyadenosine,3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxycytidine and3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyguanosine nucleosideintermediates. Treatment of the blocked intermediate as per theprocedure of Example 5 or as per the procedure of Preparation example 28of European Patent Application 0 381 335 gives the corresponding5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-(methyleneamino)oxy!adenosine, compound 27;5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-(methyleneamino)oxy!cytidine, compound 28; and5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-(methyleneamino)oxy!guanosine, compound 29.

EXAMPLE 31 3'-O-(t-Butyldiphenylsilyl)thymidine-6'-Aldehyde, 31

The title compound is prepared by homologation of the above described3'-O-(t-butyldimethylsilyl)thymidine-5'-aldehyde (compound 5) utilizingthe procedure of Barton, et al., Tetrahedron Letters 1989, 30, 4969. The5'-aldehyde, compound 5, is treated via a Witig reaction with(methoxymethylidene)triphenylphosphate. The resulting enol ether,compound 30, is hydrolyzed with Hg(OAc)₂, KI, H₂ O and THF according tothe procedure of Nicolaou, et al., J. Am. Chem. Soc. 1980, 102, 1404 tofurnish the compound 31.

EXAMPLE 325'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Nitrilomethylidyne)thymidylyl-(3'→5')-3'-O-(t-Butyldihenylsilyl)-5'-Deoxythymidine,32

The title compound is prepared by reaction of compound 31 and compound 3in the manner of Example 28, Step 1 to furnish the dimericoligonucleoside having an oxime backbone.

EXAMPLE 33 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method B

Compound 32 when treated as per the procedure of Steps 2 and 3 ofExample 28 will also yield compound 14.

EXAMPLE 34 Methyl 3'-O-Dephosphinico-3'-O-(Methyimino)methylene!-thymidylyl-(3'→5)-3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-α/β-D-erythro-Pentofuranoside,33

Compound 23 and compound 3 are linked utilizing the procedure of Example28, Steps 1 to couple the sugar and the nucleoside via an oxime linkage.The resulting oxime linkage is then reduced utilizing the procedure ofExample 28, Step 2 to an iminomethylene linkage and this linkage, inturn, when N-alkylated via the procedure of Example 28, Step 3 willyield compound 33.

EXAMPLE 35 Acetyl 5'-Benzoyl-3'-O-Dephosphinico-3'-O-(Methyimino)-methylene!thymidylyl-(3'.fwdarw.5)-3'-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-α/β-D-erythro-Pentofuranoside,34

Compound 33 will be treated with benzoyl chloride according to theprocedure of Jenkins, et al., Synthetic Procedures in Nucleic AcidChemistry, Zorbach and Tipson, Ed., Vol. 1, John Wiley & Sons, Pg. 149,to benzoylate the free 5'-hydroxyl of compound 33 which is hydrolyzedand acylated in situ according to the procedure of Baud, et. al,Tetrahedron Letters 1990, 31, 4437 to yield compound 34.

EXAMPLE 36 5'-Benzoyl-3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,35

Compound 34 is reacted with silylated thymine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene to yield5'-O-benzoyl-3'-O-dephosphinico-3'-O-(methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine,compound 35 as an anomeric mixture.

EXAMPLE 37 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method C

Compound 35 when treated with methanolic ammonia will also yieldcompound 14. Further treatment as per the procedure of Example 9 willyield the fully deblocked dimer, from which anomerically pure compound15 will be isolated by chromatography.

EXAMPLE 38 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxyadenosine,36

Compound 34 is reacted with silylated adenine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield 3'-O-dephosphinico-3'-O-(methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyadenosine,36.

EXAMPLE 39 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxycytidine37

Compound 34 is reacted with silylated cytosine as per the procedure ofBaud, et al., Tetrahedron Letters, 1990 31, 4437, utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield 3'-O-dephosphinico-3'-O-(methylimino)-methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxycytidine,37.

EXAMPLE 40 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxyguanosine38

Compound 34 is reacted with silylated guanine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield 3'-O-dephosphinico-3'-O-(methylimino)-methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyguanosine,38.

EXAMPLE 41 A-(3'→5')-T; A-(3'→5')-A; A-(3'→5')-C; and A-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoadenosine intermediate of Example 30will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is adenine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the A-T, A-A, A-C and A-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis adenine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 42 C-(3'→5')-T; C-(3'→5')-A; C-(3'→5')-C; and C-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminocytidine intermediate of Example 30will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is cytidine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the C-T, C-A, C-C and C-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis cytosine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 43 G-(3'→5')-T; G-(3'→5')-A; G-(3'→5')-C; and G-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoguanosine intermediate of Example 30will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is guanine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the G-T, G-A, G-C and G-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis guanine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 44 Trimeric, Tetrameric, Pentameric, Hexameric And Other HigherOrder Oligonucleosides Having a Selected Nucleoside Sequence

The dimers of Examples 21, 23, 24, 25, 26, 27 and 28 are extended byreaction with the 5'-(t-butyldimethylsilyl)-3'-deoxy-3'-(methyleneamino)oxy! nucleosides, compounds 10, 27, 28 and 29, ofExamples 5 and 15 to form trimers utilizing the looping sequence ofreactions of Examples 10, 11 and 12. Iteration of this reaction sequenceloop adds a further nucleoside to the growing oligonucleoside per eachiteration of the reaction sequence loop. The reaction sequence loop ofExamples 10, 11 and 12 is repeated "n" number of times to extend theoligonucleoside to the desired "n+1" length. The final 3'-blockedoligonucleoside when treated as per the procedure of Example 9 to removethe terminal 3'-O-(t-butyldiphenylsilyl) blocking group will yield thefully deblocked oligonucleoside of the selected nucleoside sequence andlength.

EXAMPLE 45 6'-Amino-6'-Deoxy-5'-Homothymidine, 42;6'-Amino-2',6'-Di-deoxy-5'-Homoadenosine, 43;6'-Amino-2',6'-Dideoxy-5'-Homocytidine, 44; and6'-Amino-2',6'-Dideoxy-5'-Homoguanosine, 45 (Via An Intramolecular FreeRadical Reaction)

Deblocking of compound 10 is effected by treatment with Bu₄ NF in THF.The resulting compound 39 (also reported in Preparation example 4 ofEuropean Patent application 0 381 335 A1) will be iodinated upontreatment with methyltriphenoxyphosphonium iodide as per the procedureof Verheyden, et al., J. Org. Chem. 1970, 35, 2119 to furnish5'-deoxy-5'-iodo-3'-O-methyleneamino-thymidine, compound 40. Compound 40when subjected to an intramolecular free radical reaction according tothe procedure of Curran, D. P., Radical Addition Reactions, InComprehensive Organic Synthesis: Trost, B. M. and Fleming, I., Eds.,vol. 4, p 715-832, Pergamon Press, Oxford (1991), will give thecorresponding 3'-O-isoxazolidinethymidine, compound 41 which on DIBAL-Hreduction will yield 6'-amino-5'-homothymidine, compound 42 the3'-(t-butyldimethylsilyl) derivative of this compound is reported inRawson, et al., Nucleosides & Nucleotides 1990, 9, 89!.

When reacted in a like manner compounds 27, 28 and 29 will give6'-amino-5'-homoadenosine, compound 43; 6'-amino-5'-homocytidine,compound 44; and 6'-amino-5'-homoguanosine, compound 45.

EXAMPLE 46 3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-C-Allylthymidine 46

A stirred solution of3'-O-(t-butyldiphenylsilyl)-5'-deoxy-5'-iododthymidine (12, 1.77 g, 3mmol), allytributyltin (2.97 g, 9 mmol) and AIBN (0.54 g, 3.3 mmol) indry toluene (30 ml) was degassed completely and heated at 65° C. for 6hr. The solution was cooled and concentrated under vacuo. The residuewas purified by silica gel column chromatography and on elution withhexanes:EtOAc (1:1, v/v) furnished the title compound as homogeneousmaterial. Appropriate fractions were pooled and evaporated to furnish46, 0.75 g of a white foam, 50% yield. The structure was confirmed by ¹H NMR.

EXAMPLE 47 3'-O-(t-Butyldiphenylsilyl)-5-Deoxy-7'-C-Aldehydothymidine 47

A solution of 46 (1 mmol), OsO₄ (0.1 mmol) and n-methylmorpholine oxide(2 mmol) in diethyl ether (4 ml) and water (2 ml) are stirred for 18 hrat room temperature. A solution of NaIO₄ (3 ml) is added and thesolution further stirred for 12 hr. The aqueous layer is extracted withdiethyl ether. Evaporation of the organic layer will give the crudealdehyde 47.

EXAMPLE 48N3-Benzoyl-1-(5'-O-Dimethoxytrityl-3'-O-Trifluoromethyl-sulfonyl-threo-Pentofuranosyl)thymine,50

The method of Horwitz, et al., J. Org. Chem. 1964, 29, 2076 will beutilized to prepare the title compound withthreo-3'-O-trifluoromethanesulfonate. Also, reaction conditions ofFleet, et al., Tetrahedron 1988, 44, 625, will furnish a 3'-leavinggroup in the threo configuration.

EXAMPLE 49 6'-O-Phthalimido-5'-Homothymidine, 52

To a stirred mixture of 5'-homothymidine Etzold, et al., ChemicalCommunications 1968, 422! (51, 1.28. g, 5 mmol), N-hydroxyphthalimide(1.09 g, 6.6 mmol) and triphenylphosphine (1.75 g, 6.6 mmol) in dry DMF(25 ml) will be added diisopropylazodicarboxylate (1.5 ml, 7.5 mmol)over a period of 30 min at 0° C. The stirring is continued for 12 hr atroom temperature. The solvent is evaporated under vacuo and the residueis washed with diethyl ether (2×50 ml). The residue will then besuspended in hot EtOH (50 ml), cooled and filtered to give the titlecompound 52.

EXAMPLE 50 6'-O-Phthalimido-3'-O-(t-Butyldiphenylsilyl)-Homothymidine 53

Compound 52 will be treated with t-butyldiphenylchlorosilane in pyridineand imidazole in a standard manner to afford the title compound 53.

EXAMPLE 51 6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-5'-Homothymidine, 54

To a stirred solution of compound 53 in dry CH₂ Cl₂ is addedmethylhydrazine (3 mmol) under anhydrous conditions at room temperature.The solution is stirred for 12 hr, cooled (0° C.) and filtered. Theprecipitate will be washed with CH₂ Cl₂ and the combined filtrates willbe concentrated. The residue is purified by flash column chromatography(silica gel, 20 g). Elution with CH₂ Cl₂ :MeOH, 9:1, v/v) will furnishthe title compound 54.

EXAMPLE 523'-De(oxophosphinico)-3'-(iminooxymethylene)-5'-Tritylthymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,55

6'-O-Amino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine, 54, isconverted to the corresponding urethane with ethyl chloroformate (CH₂Cl₂ -saturated NaHCO₃) utilizing the stereospecific conditions of Yang,et al., J. Am. Chem. Soc. 1991, 113, 4715. The residue of this reactionwill then be stirred in CH₂ Cl₂ with compound 50. The products are thenconcentrated in vacuo to yield the dimeric oligonucleoside, compound 55.

EXAMPLE 53 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-5'-Tritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,56

Compound 55 will be N-alkylated as per the conditions of Step 3 ofExample 4 to yield the N-alkylate iminooxymethylene linked dimericoligonucleoside 56.

EXAMPLE 54 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-5'-Dimethoxytritylthymidylyl-(3'→5')-5'-Deoxythymidine,57

The 5'-O-trityl and the 3'-O-(t-butyldiphenylsilyl) protecting groups ofcompound 56 will be removed by treatment with trifluoroacetic acid andthe residue dimethoxytritylated as per the procedure of Sproat, B. S.and Lamond, A. I., 2'-O-Methyloligoribonucleotides: Synthesis andApplications, oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, pg. 55 (1991), to give the title compound.

EXAMPLE 55 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-(B-Cyanoethoxy)-N-(diisopropyl)phosphiryl!-5'-Deoxythymidine, 58

Compound 57 (1.89 mmol) will be dissolved in anhydrous dichloromethaneunder an argon atmosphere. Diisopropylethylamine (0.82 ml, 4.66 mmol) isadded and the reaction mixture cooled to ice temperature.Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 ml, 4.03 mmol) isadded to the reaction mixture and the reaction mixture is allowed towarm to 20° C. and stirred for 3 hr. Ethylacetate (80 ml) andtriethylamine (1 ml) are added and the solution is washed with brinesolution three times (3×25 ml). The organic phase is separated and driedover magnesium sulfate. After filtration of the solids the solvent isevaporated in vacuo at 20° C. to an oil that will then be purified bycolumn chromatography using silica and a solvent such as hexane-ethylacetate-triethylamine (50:40:1) as eluent. The fractions are thenevaporated in vacuo and the residue will be further evaporated withanhydrous pyridine (20 ml) in vacuo (1 torr) at 26° C. in the presenceof sodium hydroxide for 24 hr to yield the title compound 58.

EXAMPLE 56 5'-Amino-5'-Homothymidine, 60

5'-Amino-3'-O-(t-butyldimethylsilyl)-5'-homothymidine 59 is prepared asper Rawson, et al., Nucleosides & Nucleotides 1990, 9, 89. Thet-butyldimethylsilyl group will be removed as per the procedure of Step4 of Example 4 to give the title compound.

EXAMPLE 57 5'-Methylamino-3'-O-(t-Butyldiphenylsilyl)-5'-Homothymidine,62

Compound 60 is t-butyldiphenylsilated as per the procedure of 37 to give5'-Amino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine, compound 61,which will then be treated as per the procedure of Step 3 of Example 4alkylate the 5'-amino group to yield the title compound 62.

EXAMPLE 58 3'-Dephosphinico-3'-S-(Methylimino)methylene!-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-3'-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,64

5'-Methylamino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine 62 (1 mmol)will be added to aqueous sodium hypochloride (4 mmol) to furnish achloramide intermediate. The chloramide intermediate is cooled (OOC) andtreated with 5'-O-monomethoxytrity-3'-thiothymidine (0.9 mmol), compound63, prepared as per Cosstick, et al., Nucleic Acids Res. 1990, 18, 829.The reaction mixture is worked up utilizing the procedure of Barton, etal., J. Org. Chem. 1991, 56, 6702 and the residue will be purified bychromatography to give the title compound 64.

EXAMPLE 59 3'-Dephosphinico-3'-S-(Methylimino)methylene!-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-5'-Deoxythymidine,65

Compound 64 will be deblocked at the terminal 3' position utilizing theas per the procedure of Step 4 of Example 4 to give compound 65.

EXAMPLE 60 3'-Dephosphinico-3'-S-(Methylimino)methylene!-5'-Monoethoxytrityl-3'-Thiothymidylyl-(3'→5')-3'(β-Cyanoethoxy)-N-(diisopropyl)phosphortityl!-5'-Deoxythymidine 66

Compound 65 will be phosphitylated as per the procedure of Example 55 togive the title compound 66.

EXAMPLE 615'-O-(t-Butyldimethylsilyl)-3'-De(oxyphosphinico)-3'-(Imino-1,2-Ethanediyl)thymidylyl-(3'→5')3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,68

3'-Amino'-5'-O-(t-butyldimethylsilyl)-3'-deoxythymidine, compound 67,prepared according to Matsuda, et al., Nucleoside & Nucleotides 1990, 9,587 will be reductively coupled with compound 47 in the presence of acatalytic amount of acid as per the procedure of Magid, et. al,Tettrahedron Letters. 1990, 31, 5595, to yield the Schiff's baseintermediate that is reduced in situ to give the amino linkage of thetitle compound 68.

EXAMPLE 62 3'-De(oxyphosphinico)-3'-(Methylimino)-1,2-Ethanediyl!-thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,69

Compound 68 will be methylated and deblocked at the 5' position as perthe procedure of Step 3 of Example 4 to yield the N-alkylated5'-deblocked dimer, compound 69.

EXAMPLE 63 3'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-(Methylimino)-1,2-Ethanediyl!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,70

Compound 69 will be dimethoxytritylated as per the procedure of Sproat,B. S. and Lamond, A. I., 2'-O-Methyloligoribonucleotides: Synthesis andApplications, Oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, 1991, pg. 55.

EXAMPLE 64 3'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-(Methylimino)-1,2-Ethanediyl!thymidylyl-(3'→5')-5'-Deoxythymidine, 71

The dimethoxytritylated intermediate, compound 70 when deblocked at the3' terminus as per the procedure of Step 4 of Example 4 will givecompound 71.

EXAMPLE 653'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-E(Methylimino)-1,2-Ethanediyl!thymidylyl-(3'→5')-3'-(B-Cyanoethoxy)-N-(diisopropyl)phosphiryl!-5'-Deoxythymidine, 72

Compound 71 will be phosphitylated as per the procedure of Example 55 togive the title compound 72.

EXAMPLE 66 2'-O-Methylhomoadenosine, 74

Homoadenosine, 73, prepared as per the procedure of Kappler, F. andHampton, A., Nucleic Acid Chemistry, Part 4, Ed. L. B. Townsend and R.S. Tipson, Wiley-Interscience Publication, 1991, pg. 240, will beblocked across its 3' and 5' hydroxyl groups with a TIPS, i.e.,tetraisopropylsilyl, blocking group followed by alkylation as per theprocedures described in U.S. patent applications 566,977, filed Aug. 13,1990 and PCT/US91/05720, filed Aug. 12, 1991. Removal of the TIPS groupwith tetra-n-butylammonium fluoride as per the procedure of Step 4 ofExample 4 will yield the title compound 74.

EXAMPLE 67 6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-5'-Homoadenosine, 75

Compound 74 will be treated as per the procedures of Examples 36, 37 and38 to yield the title compound 75.

EXAMPLE 683'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-2'-O-Methyladenosine,76

Compound 75 will be treated and reacted with compound 50 as per theprocedure of Example 65 to yield the title compound 76.

EXAMPLE 69 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl!-5'-Deoxy-2'-O-Methyladenosine,77

Compound 76 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 77.

EXAMPLE 706'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-2'-Deoxy-5'-Homoaristeromycin, 79

(-)-2'-Deoxy-5'-homoaristeromycin, compound 78, (the carbocyclic analogof 5'-homo-2'-deoxyadenosine) is prepared as per the procedure of Jones,et al., J. Chem. Soc. Perkin Trans. 1988, 1, 2927. Compound 78 will betreated as per the procedure of Examples 36, 37 and 38 to yield the6'-O-amino-3'-blocked carbocyclic analog of 5'-homo-2'-deoxyadenosine,compound 79.

EXAMPLE 713'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-2',5'-Dideoxyaristeromycin,80

Compound 79 will be treated and reacted with compound 50 as per theprocedure of Example 65 to yield the title compound 80.

EXAMPLE 72 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl!-2',5'-Dideoxyaristeromycin,81

Compound 80 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 81.

EXAMPLE 73 6'-O-Amino-2'-O-Butyl-5'-Homoaristeromycin, 82

(-)-5'-Homoaristeromycin, compound 78, will be blocked with a TIPSgroup, alkylated and deblocked as per the procedure of Example 70 toyield compound 82.

EXAMPLE 746'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-2'-O-Butyl-5'-Homoaristeromycin,83

Compound 82 will be treated as per the procedures of Examples 36, 37 and38 to yield the title compound 83.

EXAMPLE 753'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-2'-O-Butyl-5'-Deoxyaristeromycin,84

Compound 83 will be treated and reacted with compound 50 as per theprocedure of Example 65 to yield the title compound 84.

EXAMPLE 76 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl!-2'-O-Butyl-5'-Deoxyaristeromycin,85

Compound 84 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 85.

EXAMPLE 77 (+)-1-(1R,3S,4S)-3-Azido-5-Dimethoxytrityl-4-(Hydroxymethyl)-Cyclopentyl!-5-Methyl-2,4-(1H,3H)-Pyrimidindione,87

(+)-1-1R,3S,4S)-3-Azido-4-(hydroxymethyl)-cyclopentyl!-5-methyl-2,4-(1H,3H)-pyrimidindione,compound 86, prepared as per the procedure of Bodenteich, et al.,Tetrahedron Letters 1987, 28, 5311, will be dimethoxytritylatedutilizing dimethoxytrityl chloride in pyridine at room temperature togive the title compound 87.

EXAMPLE 78 (+)-1-(1R,3S,4S)-3-Amino-4-(Dimethoxytrityloxymethyl)-Cyclopentyl!-5-Methyl-2,4-(1H,3H)-Pyrimidindione,88

Compound 87 will be reduced with Ph₃ P in pyridine at room temperatureas per the procedure of Hronowski, et al., J. Chem. Soc., Chem . Commun.1990, 1547, to give the carbocyclic analog of3'-amino-5'-dimethoxytrityl thymidine, compound 88.

EXAMPLE 79 1-{(1R,3S,4S)-3-Imino-2-(5'-Deoxythymidylyl-5'-yl)-1,2-Ethanediyl!-4-(Dimethoxtrityloxymethyl)-Cyclopentyl}-5-Methyl-2,4-(1H,3H)-Pyrimidindione,89

Compound 88 will be reacted with compound 47 as per the procedure ofExample 74 to yield the title compound 89.

EXAMPLE 80 Synthesis Of Oligonucleotides Using A DNA Synthesizer

Solid support oligonucleotide and "oligonucleotide like" syntheses areperformed on an Applied Biosystems 380 B or 394 DNA synthesizerfollowing standard phosphoramidite protocols and cycles using reagentssupplied by the manufacture. The oligonucleotides are normallysynthesized in either a 10 μmol scale or a 3×1 μmol scale in the"Trityl-On" mode. Standard deprotection conditions (30% NH₄ OH, 55° C.,16 hr) are employed. HPLC is performed on a Waters 600E instrumentequipped with a model 991 detector. For analytical chromatography, thefollowing reverse phase HPLC conditions are employed: Hamilton PRP-1column (15×2.5 cm); solvent A: 50 mm TEAA, pH 7.0; solvent B: 45 mm TEAAwith 80% CH₃ CN; flow rate: 1.5 ml/min; gradient: 5% B for the first 5minutes, linear (1%) increase in B every minute thereafter. Forpreparative purposes, the following reverse phase HPLC conditions areemployed: Waters DELTA-PAK Waters Delta-Pak C₄ 15 μm, 300A, 25×100 mmcolumn equipped with a guard column of thne same material; column flowrate: 5 ml/min; gradient: 5% B for the first 10 minutes, linear 1%increase for every minute thereafter. Following HPLC purification,oligonucleotides are detritylated and further purified by size exclusionusing a SEPHADEX G-25 column.

EXAMPLE 81 Higher Order Mixed Oligonucleosides-Oligonucleosides andMixed Oligonucleosides-Oligonucleotides

A. Solution Phase Synthesis Of 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-3'-Phosphorothioate-Thymidylyl-(3'→5')-3'-De(oxyphosphinico)-3'-(Methylimino)-1,2-Ethanediyl!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,90, A Mixed oligonucleoside-Oligonucleotide-Oligonucleoside PolymerIncorporating A Nucleotide Linkage Flanked At Its 5' Terminus By A3'-De(oxophosphinico)-3'- Methyl(iminooxymethylene)! LinkedOligonucleoside Dimer and At Its 3' Terminus By A3'-De(oxyphosphinico)-3'- (Methylimino)-1,2-Ethanediyl! LinkedOligonucleoside Dimer

A mixed oligonucleoside-oligonucleotide-oligonucleoside having a3'-de(oxophosphinico)-3'- methyl(iminooxymethylene)! linkedoligonucleoside dimer and a 3'-de(oxyphosphinico)-3'-(methylimino)-1,2-ethanediyl! linked oligonucleoside dimer coupledtogether via a phosphorothioate nucleotide linkage will be prepared byreacting compound 58, compound 70 and tetrazole in anhydrousacetonitrile under argon. The coupling reaction will be allowed toproceed to completion followed by treatment with Beaucage reagent andammonium hydroxide removal of the dimethoxytrityl blocking groupaccording to the procedure of Zon, G. and Stec, W. J., Phosphorothioateoligonucleotides, Oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, pg. 87 (1991). The 3' blocking group will thenbe removed as per the procedure of Step 3 of Example 4 and the productpurified by HPLC to yield the title compound 90, wherein utilizing thestructure of FIG. 18, T₃ and T₅ are OH, D is S, E is OH, X is H, Q is O,r is 0 and q is 2; and for each q, i.e., q₁ and q₂, n and p are 1 ineach instance; and for q₁, m is 1; and for q₂, m is 0; and Bxj and Bxiare thymine.

B. Solid Support Synthesis Of 3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-(3'→5')-P-Thymidylyl-3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-(3'→5')-Thymidylyl-(3'→5')-P-Thymidylyl-3'-De(oxophosphinico)-3'-Methyl(iminooxymethylene)!-(3'→5')-Thymidylyl-(3'→5')-P-2'-Deoxycytidine,91, A Mixed Oligonucleotide-Oligonucleoside Polymer Incorporating3'-De(oxophosphinico)-3'- Methyl(iminooxymethylene)! LinkedOligonucleoside Dimers Flanked By Conventional Linked Nucleotides

The dimeric oligonucleoside 58 will be utilized as building block unitsin a conventional oligonucleotide solid support synthesis as per theprocedure of Example 80. For the purpose of illustration a polymerincorporating seven nucleosides is described. A first unit of thedimeric oligonucleoside 58 will be coupled to a first cytidinenucleoside tethered to a solid support via its 3' hydroxyl group andhaving a free 5' hydroxyl group. After attachment of the first unit ofcompound 58 to the support, the 5'-dimethoxytrityl group of that firstcompound 58 unit will be removed in the normal manner. A second compound58 unit will then be coupled via itsβ-cyanoethyl-N-diisopropylphosphiryl group to the first compound 58 unitusing normal phosphoramidate chemistry. This forms a conventionalphosphodiester bond between the first and second compound 58 units andelongates the polymer by two nucleosides (or one oligonucleoside dimerunit). The dimethoxytrityl blocking group from the second compound 58unit will be removed in the normal manner and the polymer elongated by afurther dimeric unit of compound 58. As with addition of the first andsecond dimeric units, the third unit of compound 58 is coupled to thesecond via conventional phosphoramidite procedures. The addition of thethird unit of compound 58 completes the desired length and basesequence. This polymer has a backbone of alternating normalphosphodiester linkages and the methyl-(iminooxymethylene) linkages ofcompound 58. The 5' terminal dimethoxytrityl group of the third compound58 unit will be removed in the normal manner followed by release of thepolymer from the solid support, also in the normal manner. Purificationof the polymer will be achieved by HPLC to yield compound 91 wherein,utilizing the structure of Figure, T₃ and T₅ are OH, D is O, E is OH, Xis H, Q is O , r is 1 and for the seven nucleoside polymer described, qis 3; and for each q, i.e., q₁, q₂ and q₃, n and p are 1 in eachinstances; and for q₁ and q₂, m is 1; and for q₃, m is 0; and Bxk iscytosine; and each BxJ and Bxi is thymine.

EXAMPLE 823'-O-Dephosphinico-3'-O-(Methylimino)thymidylyl-(3'→5')-5'-Deoxythymidine,9

Step 1

3'-O-Amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 1.85 g, 5 mmol),3'-O-(t-butyldimethylsilyl)thymidine-5'-aldehyde 5, 2.39 g, 5 mmol;freshly prepared by following the method of Camarasa, et al.,Nucleosides and Nucleotides 1990, 9, 533! and AcOH (0.25 ml) werestirred together in CH₂ Cl₂ (50 ml) solution at room temperature for 2h. The products were then concentrated under reduced pressure to givethe intermediate oxime linked dimer, compound 6.

Step 2

The residue obtained from Step 1 was dissolved in AcOH (25 ml). NaCNBH₃(1.55 g, 25 mmol, in 3-portions) was added to the stirred AcOH solutionat room temperature. The solution was stirred for 30 min to give theintermediate imine linked dimer, compound 7.

Step 3

Aqueous HCHO (20%, 2 ml, 66 mmol) and additional NaCNBH₃ (1.55 g, 25mmol, in 3-portions) was added to the stirred reaction mixture of Step 2at room temperature. After 2 h, the solution was diluted with EtOH (100ml), and resulting suspension was evaporated under reduced pressure. Theresidue was dissolved in CH₂ Cl₂ (150 ml) and then washed successivelywith 0.1M HCl (100 ml), saturated aqueous NaHCO₃ (100 ml), and water(2×50 ml). The dried (MgSO₄) CH₂ Cl₂ solution was evaporated to givecrude methylated imine linked dimer 8.

Step 4

The residue from Step 3 was dissolved in the THF (30 ml) and a solutionof (Bu)₄ NF (1M in THF, 10 ml) was added while stirring at roomtemperature. After 1 h, the reaction mixture was evaporated underreduced pressure and the residue was purified by short columnchromatography. The appropriate fractions, which eluted with CH₂ Cl₂:MeOH (8:2, v/v) were pooled and evaporated to give compound 9 as a foam(0.74 g, 30%). ¹ H NMR (Me₂ SO-d₆) δ 1.78 (s, 6, 2CH₃), 2.10 (m, 4,2'CH₂), 2.5 (s, 3, N--CH₃), 2.8 (m, 2, 5'-N--CH₂), 3.6-4.08 (5m, 6, 5'CH₂, 4' CH, 3' CH), 4.75 and 5.3 (2 br s, 2, 3' and 5' OH), 6.02 (d, 1,1'H), 6.1 (t, 1, 1'H), 7.4 and 7.45 (2s, 2, 2C₆ H), 11.3 (br s, 2, NH).

EXAMPLE 83 5'-O-(t-Butyldimethylsilyl)-3'-Deoxy-3'-(Methyleneamino)-oxy!thymidine, 10

A solution of HCHO (20% aqueous, 1 ml) was added dropwise to a stirredsolution of 3'-O-amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 7.42 g,20 mmol) in dry MeOH (400 ml) at room temperature. After 6 h, anotherportion of HCHO (20% aqueous, 1.5 ml) was added and stirring continuedfor 16 h. The resulting solution was evaporated under reduced pressure,and the residue was purified by chromatography on silica gel to givecompound 10 (7.25 g, 95%) as clear foam. ¹ H NMR (CDCl₃) δ 0.1 s, 3,(CH₃)₂ !, 0.9 s, 9, (CH₃)₃ !, 1.9 (s, 3, CH₃), 2.25-2.72 (m, 2, 2' CH₂),3.85-4.15 (2m, 3, 5' CH₂, 4' H), 4.85 (m, 1, 3'H), 6.25 (dd, 1, 1'H),6.5 and 6.95 (2d, 2, N═CH₂), 7.43 (s, 1, (6H), 9.2 (br s, 1 NH).

EXAMPLE 84 3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-Iodothymidine 12

To a stirred solution of 3'-O-(t-butyldiphenylsilyl)thymidine 11, 10.0g, 20.83 mmol, prepared according to the procedure of Koster, et al.,Tet. Letts. 1982, 26, 2641! in dry DMF (375 ml) was addedmethyltriphenoxyphosphonium iodide (12.12 g, 30 mmol) under argon atroom temperature. The solution was stirred for 16 h. The DMF was removedunder reduced pressure and the residue was dissolved in CH₂ Cl₂ (500ml). The organic layer was washed with 20% aqueous Na₂ S₂ O₃ (200 ml),water (2×200 ml) and dried (MgSO₄). The solvent was evaporated and theresidue was purified by silica gel chromatography. Elution with Et₂ O:Hexanes (1:1, v/v), pooling of appropriate fractions and concentrationfurnished compound 12 as white power (7.87 g, 64%, mp 142° C.). Anal.Calcd. for C₂₆ H₃₁ N₂ O₄ SiI: C, 52.88; H, 5.29; N, 4.74; I, 21.33.Found: C,52.86; H, 5.21; N, 4.66; I, 21.54.

EXAMPLE 855'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,13

A stirred solution of 5'-O-(t-butyldimethylsilyl)-3'-deoxy-3'-(methyleneamino)oxy!thymidine (10, 1.62 g, 4.23 mmol),3'-O-(t-butyldiphenylsilyl)-5'-deoxy-5'-iodothymidine (12, 2.5 g, 4.23mmol), bis(trimethylstannyl)benzopinacolate 4.84 g, 8.46 mmol, preparedaccording to the method of Hillgartner, et al., Liebigs Ann. Chem. 1975,586! in dry benzene (9 ml) was carefully degassed 3-times (flushed withargon) and heated at 80° C. for 8 h. The reaction mixture was cooled andconcentrated under reduced pressure and the residue was purified bysilica gel chromatography. The appropriate fractions, which were elutedwith CH₂ Cl₂ :MeOH (97:3, v/v), were pooled and concentrated to givedimeric oligonucleoside, compound 13 (1.25 g, 35%) as white foam. ¹ HNMR (CDCl₃) δ 0.09 and 0.13 2s, 6, (CH₃)₂ !, 0.89 and 1.06 2s, 9, (CH₃)₃!, 1.07 and 1.08 2s, 9, (CH₃)₃ !, 1.87, and 1.90 (2s, 6, 2 CH₃), 5.74(br s, 1, NH), 6.20-6.31 (2m, 2, 2 1'H), 6.88 (s, 1, C₆ H), 10.33 and10.36 (2 br s, 2, 2NH) and other protons.

EXAMPLE 86 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method A

Compound 13 was treated as per the procedure of Step 3 of Example 82 tosimultaneously N-alkylate the imino nitrogen and deblock the 5' silylblocking group of the 5' nucleoside of the dimer to yield compound 14 asa foam. ¹ H NMR (CDCl₃) δ 1.07 (s, 9, (CH₃)₃), 1.85 and 1.88 (2s, 6,2CH₃), 2.56 (s, 3, N--CH₃), 4.77 (br s, 1, 5' OH), 6.1 and 6.2 (2m, 2,1'H), 7.4 and 7.62 (2m, 10, Ph H), 9.05 (br s, 2, 2 NH), and otherprotons.

EXAMPLE 87 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-5'-Deoxythymidine, 15

The 3'-O-(t-butyldiphenylsilyl) blocking group of compound 14 is removedas per the procedure of Step 4 of Example 82 to yield the fullydeblocked dimeric oligonucleoside, compound 15.

EXAMPLE 88 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!-5'-Iodo-5'-Deoxythymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,16

Compound 14 is treated as per the procedure of Example 84 to yield thetitle dimeric oligonucleoside, compound 16, having a reactive iodofunctionality at the terminal 5' position and a blocking group remainingat the 3' position.

EXAMPLE 895'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-(Methyimino)methylene!-5'-Deoxythymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,17

Compound 16 is reacted with compound 10 utilizing the conditions ofExample 85 to extend the oligonucleoside to yield the trimericoligonucleoside, compound 17.

EXAMPLE 90 3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-(Methyimino)methylene!-5'-Deoxythymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,18

Compound 17 when reacted as per the conditions of Example 86 willundergo N-alkylation to the trimeric oligonucleoside and will be deblockat the 5' position to yield compound 18, wherein n=2.

EXAMPLE 91 3'-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-(Methyimino)methylene!-5'-Deoxythymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-(Methyimino)methylene!-5'-Deoxythymidylyl-(3'.fwdarw.5')-5'-Deoxythymidine,20

The sequence of Examples 87, 88, and 89 is repeated for the addition ofa further nucleoside to extend the oligonucleoside to a tetramer,compound 19. The tetrameric oligonucleoside 19 is then treated as perthe procedure of Example 87 to remove the terminal 3' silyl blockinggroup yielding the fully deblocked tetrameric oligonucleoside, compound20.

EXAMPLE 92 Dimer synthesis

5'-Benzoyl-3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,35

Compound 34 is reacted with silylated thymine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene to yield5'-O-benzoyl-3'-O-dephosphinico-3'-O-(methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine,compound 35 as an anomeric mixture.

3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-adenosine,36

Compound 34 is reacted with silylated adenine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield 3'-O-dephosphinico-3'-O-(methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyadenosine,36.

3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxycytidine37

Compound 34 is reacted with silylated cytosine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield 3'-O-dephosphinico-3'-O-(methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxycytidine,37.

3'-O-Dephosphinico-3'-O-(Methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxyguanosine38

Compound 34 is reacted with silylated guanine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield 3'-O-dephosphinico-3'-O-(methylimino)methylene!thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyguanosine,38.

A-(3'→5')-T; A-(3'→5')-A; A-(3'→5')-C; and A-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 44 and 92, the5'-(t-butyldimethylsilyl)-3'-O-aminoadenosine intermediate of Example 89will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is adenine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 93, 80, 81 and 95 to yield the A-T, A-A, A-C and A-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis adenine and Bxj is thymine, adenine, cytosine and guanine,respectively.

C-(3'→5')-T; C-(3'→5')-A; C-(3'→5')-C; and C-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 44 and 92, the5'-(t-butyldimethylsilyl)-3'-O-aminocytidine intermediate of Example 89will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is cytidine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 93, 80, 81 and 95 to yield the C-T, C-A, C-C and C-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis cytosine and Bxj is thymine, adenine, cytosine and guanine,respectively.

G-(3'→5')-T; G-(3'→5')-A; G-(3'→5')-C; and G-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 44 and 92, the5'-(t-butyldimethylsilyl)-3'-O-aminoguanosine intermediate of Example 89will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is guanine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 93, 80, 81 and 95 to yield the G-T, G-A, G-C and G-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis guanine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 935'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-Hydroxythymidine,48

Utilizing the procedure of Hanamoto, et al., Tet. Letts. 1991, 32, 3555,SmI₂ (0.1 mmol) in THF (3 ml) is added to a mixture of compound 5 andcompound 10 in HMPA (0.5 ml) with stirring. The mixture will be stirredat room temperature for about 15 mins to form the adduct (as detected bythe fading color). The solvent will be removed and the residue purifiedby column chromatography to give the dimeric oligonucleoside 48.

EXAMPLE 94 3'-O-Dephosphinico-3'-O-N-(Morpholin-2-yl)!thymidylyl-(3'→4')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-Demethylenethymidine,49

Utilizing the modification of Lim, M.-I. and Pan, Y.-G., Book ofAbstracts, 203 ACS national Meeting, San Francisco, Calif., Apr. 5-10,1992, of the procedure of Hill, et al., J. Chem. Soc. 1964, 3709, thedimeric oligonucleoside of Example 93 (compound 48, 1 equiv.) will betreated with chloroacetyl chloride in acetone to form an adduct with theamino group of the linkage. Further treatment with K₂ CO₃ (1.2 equiv.)in DMSO at elevated temperature will cyclize the adduct to the hydroxylgroup of the linkage to form a 5-oxomorpholino adduct with the linkage.The oxomorpholino adduct is then reduced with BH₃ -THF under reflux toyield the dimer linked via an -O- N-(morpholin-2-yl)!-linkage, compound49.

EXAMPLE 95 General method for 3'-deoxy-3'-iodo nucleosides

The preparation of 3'-deoxy-3'-iodo-5'-O-tritylthymidine has beendescribed by Verhyden, et al., J. Org. Chem. 1970, 35, 2868. In ananalogous manner, 2',3'-dideoxy-3'-iodo-5'-O-trityluridine, cytidine,adenosine and guanosine will be prepared.

EXAMPLE 96 Synthesis of Bifunctional Nucleosides

5'-Deoxy-5'iodo-3'-O-phthalimidothymidine, 102

Treatment of 3'-O-phthalimidothymidine with methyltriphenoxyphosphoniumiodide (Example 84) furnished 48% of 102; m.p. 145°-146° C.; Anal.Calcd. for C₁₈ H₁₆ N₃ O₆ I: C, 43.48; H, 3.24; N, 8.48; I, 25.52. Found:C, 43.75; H, 3.34; N, 8.38; I, 25.59. ¹ H NMR (CDCl₃) δ 8.01 (s, 3, C₅CH₃), 2.58-2.67 (m, 2, 2'CH₂), 3.54-3.78 (m, 2, 5' CH₂), 4.30-4.34 (m,1, 4'H), 5.01-5.03 (m, 1, 3'H), 6.38 (dd, 1, 1'H), 7.77-7.78 (m, 6, C₅ Hand ArH), 8.69 (br s, 1, NH).

3'-deoxy-3'-iodo-5'-O-phthalimidothymidine, 109

Treatment of 5'-O-phthalimidothymidine (Example 6) withmethyltriphenoxyphosphoniumiodide in an analogous manner gave 43% of109; m.p. 13° (decomposes); Anal. Calcd. for C₁₈ H₁₆ N₃ O₆ I:C, 43.75;H, 3.24; N, 8.45; I, 25.52. Found: C, 54.82, H, 3.24; N, 8.45; I, 25.18.¹ H NMR (CDCl₃) δ 1.94 (s, 3, C5CH₃), 2.70-2.79 (m, 2, 2'CH₂), 4.53-4.56(m, 3, 5'CH₂, 3'H), 4.67 (m, 1, 4'H), 6.28 (5, 1, 1'H), 7.70 (s, 1, CH₆H), 7.71-7.90 (m, 4, ArH), 8.55 (br s, 1, NH).

EXAMPLE 97 Incorporation Of Phosphodiester Linkages

Dimeric nucleosides 117c (R'=ODMTr, R"=O-amidite) and 119b (R'=ODMTr,R"=O-amidite), and trimeric nucleoside 118d (R'=ODMTr, R"=O-amidite, L₂=L_(2a) =N--CH₃) were prepared generally in accordance with Sproat, etal., Oligonucleotides and Analogs A Practical Approach, Eckstein, ed.,1RL Press, 1991.

3'-De(oxyphosphinico)-3'-O-(iminomethylene)-5'-dimethyoxytritylthymidylyl-(3'→5')-3'-(β-cyanoethyoxy)-N-(diisopropyl)phosphiryl!-5'-deoxythymidine 117c wasobtained as a white foam (mixture of diastereoisomer): ³¹ P NMR (CD₃ CN)δ 149.1 and 149.5 ppm; ¹ H NMR (CD₃ CN) δ 1.6 and 1.75 (2S, 6 2C₅ CH₃),2.20 (S, 3, N--CH3), 6.1 (m, 2, 1'H), 9.0 (br S, 2, NH) and otherprotons.

3'-De(oxyphosphinico)-3'- methylene(methylimino)!-thymidylyl-5'-O-(dimethytrityl)-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphilyl)thymidine 118d was obtained as white proipitate (mixture ofdiastereoisomer): ³¹ p NMR (CDCl₃) δ 149.62 and 149.11 ppm; ¹ H NMR(CDCl₃) δ 1.82 and 1.49 (2S, 6, 2C₅ CH₃), 2.58 and 2.56 (2S, 3, N--CH₃),6.16 (pseudo t, 1, T1-1'H, J=_(1'),2' =J_(1'),2" =5.8 Hz), 6.22 (pseudot, 1, T2 1'H, J=1',2'=J_(1'),2" =6.7 Hz), and other protons.

Phosphoramidites 117c, 118d, and 119b can be stored and used forcoupling by automated DNA synthesizer (e.g., ABI 380 B) as and whenrequired for specific incorporation into oligomers of therapeutic value.Other dimers of the inventions can be incorporated into oligomers in asimilar manner. This permits flexibility in converting oligonucleosidesprepared via radical coupling methodology of this invention intostandard phosphoramidites, which can be utilized as "blocks of syntheticDNA" to improve the pharmacokinetic and pharmacodynamic properties ofantisense oligomers.

EXAMPLE 98 Enzymatic Degradation

5'GCGTTTTT*TTTTTGCG3' (*=3'-CH₂ --N(CH₃)--O--CH₂ -4' linkage; 30nanomoles) was dissolved in 20 ml of buffer containing 50 mM Tris-HCl pH8.5, 14 mM MgCl₂, and 72 mM NaCl. To this solution 0.1 units ofsnake-venom phosphodiesterase (Pharmacia, Piscataway, N.J.), 23 units ofnuclease P1 (Gibco LBRL, Gaithersberg, Md.), and 24 units of calfintestinal phosphatase (Boehringer Mannheim, Indianapolis, Ind.) wasadded and the reaction mixture was incubated at 37° C. for 100 h. HPLCanalysis was carried out using a Waters model 715 automatic injector,model 600E pump, model 991 detector, and an Alltech (Alltech Associates,Inc., Deerfield, Ill.) nucleoside/nucleotide column (4.6×250 mm). Allanalyses were performed at room temperature. The solvents used were A:water and B: acetonitrile. Analysis of the nucleoside composition wasaccomplished with the following gradient: 0-5 min., 2% B (isocratic);5-20 min., 2% B to 10% B (linear); 20-40 min., 10% B to 50% B. Theintegrated area per nanomole was determined using nucleoside standards.The T*T dimer containing the N-methylaminohydroxy linkage wasquantitated by evaluation as if it were a thymidine nucleoside. Relativenucleoside ratios were calculated by converting integrated areas tomolar values and comparing all values to thymidine, which was set at itsexpected value for each oligomer.

EXAMPLE 99 3'-Deoxy-3'-C-formyl-5'-O-t-butyldiphenylsilyl-thymidine

A mixture of thymidine (400 g, and 1.65 mol), 4-dimethylaminopyridine(0.8 g, 6.5 mmol) and t-butyldiphenylchlorosilane (347.2 g, 1.26 mol) inanhydrous pyridine (3.0 lt) was stirred at room temperature for 48 hr.To the stirred reaction mixture two lots of t-butyldiphenyl chlorosilane(129.4 g, 0.47 mol and 22.7 g, 0.082 mol) were added 12 hr. apart andstirring continued for an additional 48 hr. The reaction mixture wasconcentrated under vacuum and the residue redissolved in methanol (2.5lt). The product was precipitated by pouring the reaction mixture into acold stirred ice-water (5.0 lt) suspension. The aqueous suspension wasstirred at room temperature for 3 hr. to quench traces of unreactedchlorosilane. The granular white precipitate was filtered and washedwith distilled water (5×1 lt) and air dried to furnish 876 g of5'-O-t-butyldiphenylsilyl thymidine (slightly contaminated withbissilyated product, about 5%). The impurity was removed by suspendingfinely powdered 5'-O-t-butyldiphenylsilyl thymidine in ether (600 ml)and pouring into stirred hexanes (1.5 lt).

The hexanes:ether slurry was stirred for 1 hr and filtered to furnish5'-O-t-butyldiphenylsilyl thymidine as fine white solid. The product wasfree of bissilyated impurity (judged by t1C; EtOAC:hexanes, 1:1, v/v)and on drying under vacuum furnished 718.9 g (90.7%) of5'-O-t-butyldiphenylsilyl thymidine, which was pure according tothin-layer chromatography. ¹ HNMR (DCl₃) d 1.0 (s, 9H, t BuH), 1.62 (s,3H, C5, CH₃), 2.3 (m, 2H, C2, CH₂), 2.7 (br S, 1H, 3'OH), 3.8-4.1 (m,3H, C4, H and (5'CH_(a)), 4.6 (m, 1H, 3'H), 6.45 (5, 1H, 1'H), 7.36-7.67(m, 11H, (6H and Ar H), and 9.05 (br S, 1H, NH).

To a suspension of 5'-O-i-butyldiphenylsilylthymidine (96.0 g, 0.2 mol)in dry toluene (1.1 lt) was added pyridine (19.15 g, 0.24 mol) andN-hydroxysuccimidine (4.6 g, 0.039 mol). The mixture was stirred at 55°C. under arogon while a solution of phenylchlorothioanoformate (38.28 g,0.22 mol in dry toluene, 100 ml) was added dropwise over a period of 1hr. The internal temperature of the reaction mixture rose to 70° C.while it became clear. After 24 hr, the reaction mixture pyridine (1.6g, 0.02 mol) followed by phenylchlorothionoformate (3.48 g, 0.02 mol)were added. The stirring was continued at room temperature for 24 hr.The resulting pyridinium hydrochloride salt was precipitated by additionof ether (400 ml) and filtered again. The filtrate was concentratedunder vacuum and the residue was used for subsequent radical reactionwithout any further purification.

A mixture of the 5'-O-t-butyldiphenylsilyl-3'-O-phenoxythiocarbonyl-thymidine (152.8 g, 0.24 mol),tri-n-butyltin styrene (245 g, 0.62 mol) and aza-bis-(isobutyronitrile)(5.95 g, 0.036 mol) in dry benzene (800 ml) were degassed with argon (3times) and heated at 75° C. for 8 hr while stirring. Over next 60 hr,AlBN (6×5.95 g, 0.036 mol) was added in portions to the reaction mixtureunder argon and stirring was continued at 75° C. After completion of thereaction (about 70-80 hr; detected by complete consumption of the5'-O-t-butyl diphenylsilyl-3'-O-phenoxythiocarbonyl-thymidine), thesolution was cooled to room temperature and transferred on the top of aprepacked silica gel (1 mg) column. Elution with EtOAC:Hexanes (7:5,v/v) gave the desired 3'-styryl nucleoside as homogenous material.Appropriate fractions were pooled and evaporated to furnish 67.49(49.5%) of the 3'-styryl nucleoside as an oil. ¹ HNMR (CDCl₃) d 1.1 (S,9H, Bu-H), 1.60 (S, 3H, C₅ CH₃), 2.4 (m, 2H, C_(2') CH₂) 3.25 (m, 1H,C_(3') H), 3.8 (m, 1H, C4'H), 4.15 (m, 2H, (_(5') CH₂), 6.21 (dd, 1,C_(1') H), 6.0 and 6.5 (2m, 2H, CH═CH-ph), 7.3-7.7 (m, 11H, C₆ H, and ArH), 8.8 (S, 1H, OH).

A mixture of the 3'-styryl nucleoside (2.19 g, 3.86 mmol), N-methylmorpholine-N-oxide (0.68 g, 5.6 mmol), OsO₄ (3.9 ml of 2.5% solution int-BuOH, 0.38 mmol) in dioxane: water (30 ml, 2:1) was stirred at roomtemperature. The reaction mixture was protected from light and stirredfor 1 hr. To the dark colored reaction mixture NaIO₄ (1.82 g, 8.5 mmol)in water (8 ml) was added in one portion and stirring continued for 3hr. After completion of the reaction, the reaction mixture was dilutedwith EtOAC (100 ml) and extracted with saturated NaCl solution (3×60ml). The organic layer was dried (MgSO₄) and concentrated to furnishoily residue. The residue was purified by silica gel columnchromatography to furnish 0.94 g (50%) of5'-O-t-butyldiphenylsilyl-3'-c-formyl-thymidine as white foam. ¹ HNMR(CDCl₃) d 1.1 (s, 9H, t-BuH), 1.61 (S, 3H, C₅ CH₃), 2.3 and 2.75 (2m,2H, C2, CH₂), 3.4 (m, 1H, C_(3') H), 4.0 (m, 2H, C_(5') CH₂), 4.35 (m,1H, C_(4') H), 6.11 (t, 1, C_(1') H), 7.26-7.67 (m, 11H, C₆ H, Ar H),8.2 (brS, 1H, NH), and 9.70 (s, 1H, CHO).

Evaluation Procedure 1--Nuclease Resistance

A. Evaluation of the resistance of oligonucleotide-mimickingmacromolecules to serum and cytoplasmic nucleases.

Oligonucleotide-mimicking macromolecules of the invention can beassessed for their resistance to serum nucleases by incubation of theoligonucleotide-mimicking macromolecules in media containing variousconcentrations of fetal calf serum or adult human serum. Labeledoligonucleotide-mimicking macromolecules are incubated for varioustimes, treated with protease K and then analyzed by gel electrophoresison 20% polyacrylamine-urea denaturing gels and subsequentautoradiography. Autoradiograms are quantitated by laser densitometry.Based upon the location of the modified linkage and the known length ofthe oligonucleotide-mimicking macromolecules it is possible to determinethe effect on nuclease degradation by the particular modification. Forthe cytoplasmic nucleases, an HL 60 cell line can be used. Apost-mitochondrial supernatant is prepared by differentialcentrifugation and the labelled macromolecules are incubated in thissupernatant for various times. Following the incubation, macromoleculesare assessed for degradation as outlined above for serum nucleolyticdegradation. Autoradiography results are quantitated for evaluation ofthe macromolecules of the invention. It is expected that themacromolecules will be completely resistant to serum and cytoplasmicnucleases.

B. Evaluation of the resistance of oligonucleotide-mimickingmacromolecules to specific endo- and exo-nucleases.

Evaluation of the resistance of natural oligonucleotides andoligonucleotide-mimicking macromolecules of the invention to specificnucleases (ie, endonucleases, 3',5'-exo-, and 5',3'-exonucleases) can bedone to determine the exact effect of the macromolecule linkage ondegradation. The oligonucleotide-mimicking macromolecules are incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea is added andanalysis on 20% polyacrylamide gels containing urea is done. Gelproducts are visualized by staining with STAINS ALL reagent (SigmaChemical Co.). Laser densitometry is used to quantitate the extent ofdegradation. The effects of the macromolecules linkage are determinedfor specific nucleases and compared with the results obtained from theserum and cytoplasmic systems. As with the serum and cytoplasmicnucleases, it is expected that the oligonucleotide-mimickingmacromolecules of the invention will be completely resistant to endo-and exo-nucleases.

C. Nuclease Degradation Studies

It has been reported that terminal phosphorothioate andmethylphosphonate modifications stabilize an oligonucleotide to 3' and5' exonucleases such as snake venom phosphodiesteraser, spleenphosphodiesterase, calf serum, and cell media (see, e.g., Nuclec AcidsRes. 1991, 19, 747 and 5473). The novel backbones of this inventionconfer similar or sometimes better protection from enzymaticexonucleolytic degradation. The inventors recently reported (J. Am.Chem. Soc. 114, 4006) that incorporation of a single 3'-CH₂--N(CH₃)--O--CH2-4' backbone at the 3' terminus of an oligomer enhancedthe half-life of modified oligomer compared to the natural unmodifiedoligomer. These oligonucleosides exhibited a significant resistance tonucleases while maintaining a high level of base pair specificity.Therefore, the following oligonucleosides were modified at theirterminal positions (3' and/or 5') with 3'-CH₂ --N(CH₃)--O--CH2-4'linkages to block exonucleolytic degradation.

    ______________________________________                                                 Oligonucleoside                                                      No.      Sequence        T 1/2 (Hours)                                        ______________________________________                                        1        TTTTTTTTTTC     0.2                                                  2        T*TT*TTTT*TT*TC 0.4 (11 mer) 11 (10-mer)                             3        T*TTTTTTTT*TC   0.4 (11-mer) 6 (10-mer)                              4        T*TT*TT*TT*TT*TC                                                                              0.4 (11-mer) 16 (10-mer)                             5        T*T*T*T         No degradation|                                                               (up to ˜60 hours)                              ______________________________________                                    

Results for Entries 2-4 showed a characteristic 3'-exonuclease-dominantdegradation pattern characterized by rapid cleavage of 3'-C residue fromall oligomers. Asid from the 3'-phosphodiester linkages, the 10-mersappear to be significantly resistant towards degradation compared to theunmodified oligomer (Entry 1). The fully modified tetramer (Entry 5),which contains no phospodiester linkage, showed complete stability up to60 houre of incubation in cell extract (see, Nucleic Acids Res. 19, 5743for experimental details). Since the phosphodiester linkage is the siteof nucleolytic attack, complete stability for the fully modifiedoligomer was expected. These results taken together suggests that anend-capped (3' and 5') oligomer containing achiral and neutral backbonewill have enhance half-life.

Procedure 2--5-Lipoxygenase Analysis and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering the macromolecule of the invention. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Such treatment is generallycontinued until either a cure is effected or a diminution in thediseased state is achieved. Long term treatment is likely for somediseases.

B. Research Reagents

The oligonucleotide-mimicking macromolecules of this invention will alsobe useful as research reagents when used to cleave or otherwise modulate5-lipoxygenase mRNA in crude cell lysates or in partially purified orwholly purified RNA preparations. This application of the invention isaccomplished, for example, by lysing cells by standard methods,optimally extracting the RNA and then treating it with a composition atconcentrations ranging, for instance, from about 100 to about 500 ng per10 Mg of total RNA in a buffer consisting, for example, of 50 mmphosphate, pH ranging from about 4-10 at a temperature from about 30° toabout 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarosegel electrophoresis and hybridization with radiolabeled DNA probes or byother standard methods.

C. Diagnostics

The oligonucleotide-mimicking macromolecules of the invention will alsobe useful in diagnostic applications, particularly for the determinationof the expression of specific mRNA species in various tissues or theexpression of abnormal or mutant RNA species. In this example, while themacromolecules target a abnormal mRNA by being designed complementary tothe abnormal sequence, they would not hybridize to normal mRNA.

Tissue samples can be homogenized, and RNA extracted by standardmethods. The crude homogenate or extract can be treated for example toeffect cleavage of the target RNA. The product can then be hybridized toa solid support which contains a bound oligonucleotide complementary toa region on the 5' side of the cleavage site. Both the normal andabnormal 5' region of the mRNA would bind to the solid support. The 3'region of the abnormal RNA, which is cleaved, would not be bound to thesupport and therefore would be separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotide-mimicking macromolecules whichinterfere with 5-lipoxygenase synthesis in these cells.

A second test system for oligonucleotide-mimicking macromolecules makesuse of the fact that 5-lipoxygenase is a "suicide" enzyme in that itinactivates itself upon reacting with substrate. Treatment ofdifferentiated HL-60 or other cells expressing 5 lipoxygenase, with 10μM A23187, a calcium ionophore, promotes translocation of 5-lipoxygenasefrom the cytosol to the membrane with subsequent activation of theenzyme. Following activation and several rounds of catalysis, the enzymebecomes catalytically inactive. Thus, treatment of the cells withcalcium ionophore inactivates endogenous 5-lipoxygenase. It takes thecells approximately 24 hours to recover from A23187 treatment asmeasured by their ability to synthesize leukotriene B₄. Macromoleculesdirected against 5-lipoxygenase can be tested for activity in two HL-60model systems using the following quantitative assays. The assays aredescribed from the most direct measurement of inhibition of5-lipoxygenase protein synthesis in intact cells to more downstreamevents such as measurement of 5-lipoxygenase activity in intact cells.

A direct effect which oligonucleotide-mimicking macromolecules can exerton intact cells and which can be easily be quantitated is specificinhibition of 5-lipoxygenase protein synthesis. To perform thistechnique, cells can be labelled with ³⁵ S-methionine (50 μCi/mL) for 2hours at 37° C. to label newly synthesized protein. Cells are extractedto solubilize total cellular proteins and 5-lipoxygenase isimmunoprecipitated with 5-lipoxygenase antibody followed by elution fromprotein A Sepharose beads. The immunoprecipitated proteins are resolvedby SDS-polyacrylamide gel electrophoresis and exposed forautoradiography. The amount of immunoprecipitated 5-lipoxygenase isquantitated by scanning densitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for quantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM ¹⁴C-arachidonic acid, 2mM ATP, 50 μM free calcium, 100 μg/mlphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NOVAPAKC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective the macromolecules of the invention at 1 μM, 10μM, and 30 μM would be as follows. Control cells oxidize 200 pmolarachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30μM of an effective oligonucleotide-mimicking macromolecule would oxidize195 μmol, 140 μmol, and 60 μmol of arachidonic acid/5 min/10⁶ cellsrespectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is usedas a standard and as the primary antigen to coat microtiter plates. 25ng of purified 5-lipoxygenase is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris•HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labelled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 meroligonucleotide-mimicking macromolecule at 1 μM, 10 μM, and 30 μM wouldbe 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10⁶ cells, respectivelywith untreated cells containing about 34 ng 5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with anoligonucleotide-mimicking macromolecule directed to the 5-LO mRNA. Cellswill be treated for 72 hours with either 1 μM, 10 μM or 30 μM of themacromolecule in the presence of 1.3% DMSO. The quantity of LTB₄produced from 5×10⁵ cells would be expected to be about 75 pg, 50 pg,and 35 pg, respectively with untreated differentiated cells producing 75pg LTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotide-mimicking macromolecules will be appliedtopically to both ears 12 to 24 hours prior to administration ofarachidonic acid to allow optimal activity of the compounds. Both earsare pretreated for 24 hours with either 0.1 μmol, 0.3 μmol, or 1.0 μmolof the macromolecule prior to challenge with arachidonic acid. Valuesare expressed as the mean for three animals per concentration.Inhibition of polymorphonuclear cell infiltration for 0.1 μmol, 0.3μmol, and 1 μmol is expected to be about 10%, 75% and 92% of controlactivity, respectively. Inhibition of edema is expected to be about 3%,58% and 90%, respectively while inhibition of leukotriene B₄ productionwould be expected to be about 15%, 79% and 99%, respectively.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A compound having structure: ##STR7## wherein: L₁--L₂ --L₃ --L₄, respectively, are CH₂ --NR₁ --NR₂ --CH₂, CH₂ --CH₂ --NR₁--NR₂, or NR₁ --NR₂ --CH₂ --CH₂ ;R₁ and R₂ are the same or different andare H; alkyl having 1 to 10 carbon atoms; alkenyl having 2 to 10 carbonatoms; alkynyl having 2 to 10 carbon atoms; aralkyl having 7 to 14carbon atoms; alicyclic; heterocyclic; a reporter molecule; or an RNAcleaving group; B_(x) is a nucleosidic base; Q is O, S, CH₂, CHF or CF₂; n is an integer greater than O; X is H, OH, C₁, to C₁₀ lower alkyl,aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloaralkyl, aminoalkylamino, polyalkylamino,substituted silyl, or an RNA cleaving group "wherein said compoundexhibits nuclease resistance and base pair binding specificity".
 2. Thecompound of claim 1 wherein L₁ --L₂ --L₃ --L₄ is CH₂ --NR₁ --NR₂ --CH₂.3. The compound of claim 1 wherein L₁ --L₂ --L₃ --L₄ is NR₁ --NR₂ --CH₂--CH₂.
 4. The compound of claim 1 wherein at least one of R₁ and R₂ isC₁ to C₁₀ alkyl.
 5. The compound of claim 1 wherein at least one of R₁and R₂ is H.
 6. The compound of claim 1 wherein at least one of R₁ andR₂ is C₁ to C₁₀ straight chain lower alkyl or C₃ to C₁₀ branched chainlower alkyl; C₂ to C₁₀ straight chain lower alkenyl or C₃ to C₁₀branched chain lower alkenyl; C₂ to C₁₀ straight chain lower alkynyl orC₃ to C₁₀ branched chain lower alkynyl.
 7. The compound of claim 1wherein at least one of R₁ and R₂ is a ¹⁴ C containing lower alkyl,lower alkenyl or lower alkynyl; or a ¹⁴ C containing C₇ to C₁₄ aralkyl.8. The compound of claim 1 wherein at least one of R₁ and R₂ is areporter molecule or an RNA cleaving group.
 9. The compound of claim 1wherein Q is O.
 10. The compound of claim 1 wherein X is H or OH.